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Sample records for active space cas

  1. Dual nuclease activity of a Cas2 protein in CRISPR-Cas subtype I-B of Leptospira interrogans.

    PubMed

    Dixit, Bhuvan; Ghosh, Karukriti Kaushik; Fernandes, Gary; Kumar, Pankaj; Gogoi, Prerana; Kumar, Manish

    2016-04-01

    Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 carries a set of cas genes associated with CRISPR-Cas subtype I-B. Herein, we report for the first time active transcription of a set of cas genes (cas1 to cas8) of L. interrogans where cas4, cas1, cas2 and cas6, cas3, cas8, cas7, cas5 are clustered together in two independent operons. As an initial step toward comprehensive understanding of CRISPR-Cas system in spirochete, the biochemical study of one of the core Leptospira Cas2 proteins (Lep_Cas2) showed nuclease activity on both DNA and RNA in a nonspecific manner. Additionally, unlike other known Cas2 proteins, Lep_Cas2 showed metal-independent RNase activity and preferential activity on RNA over DNA. These results provide insight for understanding Cas2 diversity existing in the prokaryotic adaptive immune system.

  2. Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro.

    PubMed

    Hinz, John M; Laughery, Marian F; Wyrick, John J

    2015-12-08

    During Cas9 genome editing in eukaryotic cells, the bacterial Cas9 enzyme cleaves DNA targets within chromatin. To understand how chromatin affects Cas9 targeting, we characterized Cas9 activity on nucleosome substrates in vitro. We find that Cas9 endonuclease activity is strongly inhibited when its target site is located within the nucleosome core. In contrast, the nucleosome structure does not affect Cas9 activity at a target site within the adjacent linker DNA. Analysis of target sites that partially overlap with the nucleosome edge indicates that the accessibility of the protospacer-adjacent motif (PAM) is the critical determinant of Cas9 activity on a nucleosome.

  3. Guide RNA functional modules direct Cas9 activity and orthogonality.

    PubMed

    Briner, Alexandra E; Donohoue, Paul D; Gomaa, Ahmed A; Selle, Kurt; Slorach, Euan M; Nye, Christopher H; Haurwitz, Rachel E; Beisel, Chase L; May, Andrew P; Barrangou, Rodolphe

    2014-10-23

    The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner and has been widely used for programmable genome editing. Cas9 activity is dependent on interactions with guide RNAs, and evolutionarily divergent Cas9 nucleases have been shown to work orthogonally. However, the molecular basis of selective Cas9:guide-RNA interactions is poorly understood. Here, we identify and characterize six conserved modules within native crRNA:tracrRNA duplexes and single guide RNAs (sgRNAs) that direct Cas9 endonuclease activity. We show the bulge and nexus are necessary for DNA cleavage and demonstrate that the nexus and hairpins are instrumental in defining orthogonality between systems. In contrast, the crRNA:tracrRNA complementary region can be modified or partially removed. Collectively, our results establish guide RNA features that drive DNA targeting by Cas9 and open new design and engineering avenues for CRISPR technologies.

  4. Structural plasticity and in vivo activity of Cas1 from the type I-F CRISPR-Cas system.

    PubMed

    Wilkinson, Max E; Nakatani, Yoshio; Staals, Raymond H J; Kieper, Sebastian N; Opel-Reading, Helen K; McKenzie, Rebecca E; Fineran, Peter C; Krause, Kurt L

    2016-04-15

    CRISPR-Cas systems are adaptive immune systems in prokaryotes that provide protection against viruses and other foreign DNA. In the adaptation stage, foreign DNA is integrated into CRISPR (clustered regularly interspaced short palindromic repeat) arrays as new spacers. These spacers are used in the interference stage to guide effector CRISPR associated (Cas) protein(s) to target complementary foreign invading DNA. Cas1 is the integrase enzyme that is central to the catalysis of spacer integration. There are many diverse types of CRISPR-Cas systems, including type I-F systems, which are typified by a unique Cas1-Cas2-3 adaptation complex. In the present study we characterize the Cas1 protein of the potato phytopathogen Pectobacterium atrosepticum, an important model organism for understanding spacer acquisition in type I-F CRISPR-Cas systems. We demonstrate by mutagenesis that Cas1 is essential for adaptation in vivo and requires a conserved aspartic acid residue. By X-ray crystallography, we show that although P. atrosepticum Cas1 adopts a fold conserved among other Cas1 proteins, it possesses remarkable asymmetry as a result of structural plasticity. In particular, we resolve for the first time a flexible, asymmetric loop that may be unique to type I-F Cas1 proteins, and we discuss the implications of these structural features for DNA binding and enzymatic activity.

  5. Interference activity of a minimal Type I CRISPR–Cas system from Shewanella putrefaciens

    PubMed Central

    Dwarakanath, Srivatsa; Brenzinger, Susanne; Gleditzsch, Daniel; Plagens, André; Klingl, Andreas; Thormann, Kai; Randau, Lennart

    2015-01-01

    Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs. PMID:26350210

  6. Cas9 gRNA engineering for genome editing, activation and repression.

    PubMed

    Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; Hall, Richard N; Chari, Raj; Ter-Ovanesyan, Dmitry; Qian, Jason; Pruitt, Benjamin W; Beal, Jacob; Vora, Suhani; Buchthal, Joanna; Kowal, Emma J K; Ebrahimkhani, Mohammad R; Collins, James J; Weiss, Ron; Church, George

    2015-11-01

    We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

  7. Cas9 gRNA engineering for genome editing, activation and repression

    SciTech Connect

    Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; Hall, Richard N.; Chari, Raj; Ter-Ovanesyan, Dmitry; Qian, Jason; Pruitt, Benjamin W.; Beal, Jacob; Vora, Suhani; Buchthal, Joanna; Kowal, Emma J. K.; Ebrahimkhani, Mohammad R.; Collins, James J.; Weiss, Ron; Church, George

    2015-09-07

    Here we demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

  8. Orthogonal gene knock out and activation with a catalytically active Cas9 nuclease

    PubMed Central

    Dahlman, James E.; Abudayyeh, Omar O.; Joung, Julia; Gootenberg, Jonathan S.; Zhang, Feng; Konermann, Silvana

    2015-01-01

    We have developed a CRISPR-based method that uses catalytically active Cas9 and distinct sgRNA constructs to knock out and activate different genes in the same cell. These sgRNAs, with 14 15 bp target sequences and MS2 binding loops, can activate gene expression using an active Cas9 nuclease, without inducing DSBs. We use these ‘dead RNAs’ to perform orthogonal gene knockout and transcriptional activation in human cells. PMID:26436575

  9. Cas9 gRNA engineering for genome editing, activation and repression

    PubMed Central

    Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; Hall, Richard N; Chari, Raj; Ter-Ovanesyan, Dmitry; Qian, Jason; Pruitt, Benjamin W; Beal, Jacob; Vora, Suhani; Buchthal, Joanna; Kowal, Emma J K; Ebrahimkhani, Mohammad R; Collins, James J; Weiss, Ron; Church, George

    2015-01-01

    We demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein. PMID:26344044

  10. Examination of CRISPR/Cas9 design tools and the effect of target site accessibility on Cas9 activity.

    PubMed

    Lee, Ciaran M; Davis, Timothy H; Bao, Gang

    2017-03-16

    The recent adaptation of the CRISPR/Cas9 system for targeted genome engineering has led to its widespread applications in many fields worldwide. In order to better understand the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design these studies have spawned a plethora of gRNA design tools with algorithms based solely on direct or indirect sequence features. Here we demonstrate that the predictive power of these tools is poor, suggesting that sequence features alone cannot accurately inform the cutting efficiency of a particular CRISPR/Cas9 gRNA design. Furthermore we demonstrate that DNA target site accessibility influences the activity of CRISPR/Cas9. With further optimisation we hypothesise that it will be possible to increase the predictive power of gRNA design tools by including both sequence and target site accessibility metrics. This article is protected by copyright. All rights reserved.

  11. Nuclease activity of Legionella pneumophila Cas2 promotes intracellular infection of amoebal host cells.

    PubMed

    Gunderson, Felizza F; Mallama, Celeste A; Fairbairn, Stephanie G; Cianciotto, Nicholas P

    2015-03-01

    Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain of L. pneumophila that naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts.

  12. Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

    PubMed Central

    Gunderson, Felizza F.; Mallama, Celeste A.; Fairbairn, Stephanie G.

    2014-01-01

    Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain of L. pneumophila that naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts. PMID:25547789

  13. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation.

    PubMed

    Jinek, Martin; Jiang, Fuguo; Taylor, David W; Sternberg, Samuel H; Kaya, Emine; Ma, Enbo; Anders, Carolin; Hauer, Michael; Zhou, Kaihong; Lin, Steven; Kaplan, Matias; Iavarone, Anthony T; Charpentier, Emmanuelle; Nogales, Eva; Doudna, Jennifer A

    2014-03-14

    Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

  14. Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease

    PubMed Central

    Lim, Youngbin; Bak, So Young; Sung, Keewon; Jeong, Euihwan; Lee, Seung Hwan; Kim, Jin-Soo; Bae, Sangsu; Kim, Seong Keun

    2016-01-01

    The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex. For the Cas9:gRNA complex, we identify sub-conformations of the RNA–DNA heteroduplex during R-loop expansion. Our single-molecule study indicates that the kinetics of the sub-conformations is controlled by the complementarity between crRNA and target DNA. We conclude that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system. PMID:27804953

  15. Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease.

    PubMed

    Lim, Youngbin; Bak, So Young; Sung, Keewon; Jeong, Euihwan; Lee, Seung Hwan; Kim, Jin-Soo; Bae, Sangsu; Kim, Seong Keun

    2016-11-02

    The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex. For the Cas9:gRNA complex, we identify sub-conformations of the RNA-DNA heteroduplex during R-loop expansion. Our single-molecule study indicates that the kinetics of the sub-conformations is controlled by the complementarity between crRNA and target DNA. We conclude that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system.

  16. Selection of active spaces for multiconfigurational wavefunctions

    SciTech Connect

    Keller, Sebastian; Boguslawski, Katharina; Reiher, Markus; Janowski, Tomasz; Pulay, Peter

    2015-06-28

    The efficient and accurate description of the electronic structure of strongly correlated systems is still a largely unsolved problem. The usual procedures start with a multiconfigurational (usually a Complete Active Space, CAS) wavefunction which accounts for static correlation and add dynamical correlation by perturbation theory, configuration interaction, or coupled cluster expansion. This procedure requires the correct selection of the active space. Intuitive methods are unreliable for complex systems. The inexpensive black-box unrestricted natural orbital (UNO) criterion postulates that the Unrestricted Hartree-Fock (UHF) charge natural orbitals with fractional occupancy (e.g., between 0.02 and 1.98) constitute the active space. UNOs generally approximate the CAS orbitals so well that the orbital optimization in CAS Self-Consistent Field (CASSCF) may be omitted, resulting in the inexpensive UNO-CAS method. A rigorous testing of the UNO criterion requires comparison with approximate full configuration interaction wavefunctions. This became feasible with the advent of Density Matrix Renormalization Group (DMRG) methods which can approximate highly correlated wavefunctions at affordable cost. We have compared active orbital occupancies in UNO-CAS and CASSCF calculations with DMRG in a number of strongly correlated molecules: compounds of electronegative atoms (F{sub 2}, ozone, and NO{sub 2}), polyenes, aromatic molecules (naphthalene, azulene, anthracene, and nitrobenzene), radicals (phenoxy and benzyl), diradicals (o-, m-, and p-benzyne), and transition metal compounds (nickel-acetylene and Cr{sub 2}). The UNO criterion works well in these cases. Other symmetry breaking solutions, with the possible exception of spatial symmetry, do not appear to be essential to generate the correct active space. In the case of multiple UHF solutions, the natural orbitals of the average UHF density should be used. The problems of the UNO criterion and their potential solutions

  17. Selection of active spaces for multiconfigurational wavefunctions

    NASA Astrophysics Data System (ADS)

    Keller, Sebastian; Boguslawski, Katharina; Janowski, Tomasz; Reiher, Markus; Pulay, Peter

    2015-06-01

    The efficient and accurate description of the electronic structure of strongly correlated systems is still a largely unsolved problem. The usual procedures start with a multiconfigurational (usually a Complete Active Space, CAS) wavefunction which accounts for static correlation and add dynamical correlation by perturbation theory, configuration interaction, or coupled cluster expansion. This procedure requires the correct selection of the active space. Intuitive methods are unreliable for complex systems. The inexpensive black-box unrestricted natural orbital (UNO) criterion postulates that the Unrestricted Hartree-Fock (UHF) charge natural orbitals with fractional occupancy (e.g., between 0.02 and 1.98) constitute the active space. UNOs generally approximate the CAS orbitals so well that the orbital optimization in CAS Self-Consistent Field (CASSCF) may be omitted, resulting in the inexpensive UNO-CAS method. A rigorous testing of the UNO criterion requires comparison with approximate full configuration interaction wavefunctions. This became feasible with the advent of Density Matrix Renormalization Group (DMRG) methods which can approximate highly correlated wavefunctions at affordable cost. We have compared active orbital occupancies in UNO-CAS and CASSCF calculations with DMRG in a number of strongly correlated molecules: compounds of electronegative atoms (F2, ozone, and NO2), polyenes, aromatic molecules (naphthalene, azulene, anthracene, and nitrobenzene), radicals (phenoxy and benzyl), diradicals (o-, m-, and p-benzyne), and transition metal compounds (nickel-acetylene and Cr2). The UNO criterion works well in these cases. Other symmetry breaking solutions, with the possible exception of spatial symmetry, do not appear to be essential to generate the correct active space. In the case of multiple UHF solutions, the natural orbitals of the average UHF density should be used. The problems of the UNO criterion and their potential solutions are discussed

  18. A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Cas9 endonuclease of the Type II-a clustered regularly interspersed short palindromic repeats (CRISPR), of Streptococcus pyogenes (SpCas9) has been adapted as a widely used tool for genome editing and genome engineering. Herein, we describe a gene encoding a novel Cas9 ortholog (BpsuCas9) and th...

  19. French space activities

    NASA Technical Reports Server (NTRS)

    Blanc, R.

    1982-01-01

    The four main points of research and development of space programs by France are explained. The National Center of Space Studies is discussed, listing the missions of the Center and describing the activities of the staff.

  20. A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation

    PubMed Central

    Farasat, Iman; Salis, Howard M.

    2016-01-01

    The ability to precisely modify genomes and regulate specific genes will greatly accelerate several medical and engineering applications. The CRISPR/Cas9 (Type II) system binds and cuts DNA using guide RNAs, though the variables that control its on-target and off-target activity remain poorly characterized. Here, we develop and parameterize a system-wide biophysical model of Cas9-based genome editing and gene regulation to predict how changing guide RNA sequences, DNA superhelical densities, Cas9 and crRNA expression levels, organisms and growth conditions, and experimental conditions collectively control the dynamics of dCas9-based binding and Cas9-based cleavage at all DNA sites with both canonical and non-canonical PAMs. We combine statistical thermodynamics and kinetics to model Cas9:crRNA complex formation, diffusion, site selection, reversible R-loop formation, and cleavage, using large amounts of structural, biochemical, expression, and next-generation sequencing data to determine kinetic parameters and develop free energy models. Our results identify DNA supercoiling as a novel mechanism controlling Cas9 binding. Using the model, we predict Cas9 off-target binding frequencies across the lambdaphage and human genomes, and explain why Cas9’s off-target activity can be so high. With this improved understanding, we propose several rules for designing experiments for minimizing off-target activity. We also discuss the implications for engineering dCas9-based genetic circuits. PMID:26824432

  1. Space construction activities

    NASA Technical Reports Server (NTRS)

    1991-01-01

    The Center for Space Construction at the University of Colorado at Boulder was established in 1988 as a University Space Engineering Research Center. The mission of the Center is to conduct interdisciplinary engineering research which is critical to the construction of future space structures and systems and to educate students who will have the vision and technical skills to successfully lead future space construction activities. The research activities are currently organized around two central projects: Orbital Construction and Lunar Construction. Summaries of the research projects are included.

  2. The New State of the Art: Cas9 for Gene Activation and Repression

    PubMed Central

    La Russa, Marie F.

    2015-01-01

    CRISPR-Cas9 technology has rapidly changed the landscape for how biologists and bioengineers study and manipulate the genome. Derived from the bacterial adaptive immune system, CRISPR-Cas9 has been coopted and repurposed for a variety of new functions, including the activation or repression of gene expression (termed CRISPRa or CRISPRi, respectively). This represents an exciting alternative to previously used repression or activation technologies such as RNA interference (RNAi) or the use of gene overexpression vectors. We have only just begun exploring the possibilities that CRISPR technology offers for gene regulation and the control of cell identity and behavior. In this review, we describe the recent advances of CRISPR-Cas9 technology for gene regulation and outline advantages and disadvantages of CRISPRa and CRISPRi (CRISPRa/i) relative to alternative technologies. PMID:26370509

  3. Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference

    SciTech Connect

    Beloglazova, Natalia; Petit, Pierre; Flick, Robert; Brown, Greg; Savchenko, Alexei; Yakunin, Alexander F.

    2012-03-15

    Clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas proteins represent an adaptive microbial immunity system against viruses and plasmids. Cas3 proteins have been proposed to play a key role in the CRISPR mechanism through the direct cleavage of invasive DNA. Here, we show that the Cas3 HD domain protein MJ0384 from Methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3'-5') single-stranded DNAs and RNAs, as well as 3'-flaps, splayed arms, and R-loops. The degradation of branched DNA substrates by MJ0384 is stimulated by the Cas3 helicase MJ0383 and ATP. The crystal structure of MJ0384 revealed the active site with two bound metal cations and together with site-directed mutagenesis suggested a catalytic mechanism. Our studies suggest that the Cas3 HD nucleases working together with the Cas3 helicases can completely degrade invasive DNAs through the combination of endo- and exonuclease activities.

  4. Potent and Targeted Activation of Latent HIV-1 Using the CRISPR/dCas9 Activator Complex.

    PubMed

    Saayman, Sheena M; Lazar, Daniel C; Scott, Tristan A; Hart, Jonathan R; Takahashi, Mayumi; Burnett, John C; Planelles, Vicente; Morris, Kevin V; Weinberg, Marc S

    2016-03-01

    HIV-1 provirus integration results in a persistent latently infected reservoir that is recalcitrant to combined antiretroviral therapy (cART) with lifelong treatment being the only option. The "shock and kill" strategy aims to eradicate latent HIV by reactivating proviral gene expression in the context of cART treatment. Gene-specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising single guide RNAs (sgRNAs) with a nuclease-deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). We engineered this system to target 23 sites within the long terminal repeat promoter of HIV-1 and identified a "hotspot" for activation within the viral enhancer sequence. Activating sgRNAs transcriptionally modulated the latent proviral genome across multiple different in vitro latency cell models including T cells comprising a clonally integrated mCherry-IRES-Tat (LChIT) latency system. We detected consistent and effective activation of latent virus mediated by activator sgRNAs, whereas latency reversal agents produced variable activation responses. Transcriptomic analysis revealed dCas9-VP64/sgRNAs to be highly specific, while the well-characterized chemical activator TNFα induced widespread gene dysregulation. CRISPR-mediated gene activation represents a novel system which provides enhanced efficiency and specificity in a targeted latency reactivation strategy and represents a promising approach to a "functional cure" of HIV/AIDS.

  5. Actin polymerization-dependent activation of Cas-L promotes immunological synapse stability

    PubMed Central

    Santos, Luís C; Blair, David A; Kumari, Sudha; Cammer, Michael; Iskratsch, Thomas; Herbin, Olivier; Alexandropoulos, Konstantina; Dustin, Michael L; Sheetz, Michael P

    2016-01-01

    The immunological synapse formed between a T-cell and an antigen-presenting cell is important for cell–cell communication during T-cell-mediated immune responses. Immunological synapse formation begins with stimulation of the T-cell receptor (TCR). TCR microclusters are assembled and transported to the center of the immunological synapse in an actin polymerization-dependent process. However, the physical link between TCR and actin remains elusive. Here we show that lymphocyte-specific Crk-associated substrate (Cas-L), a member of a force sensing protein family, is required for transport of TCR microclusters and for establishing synapse stability. We found that Cas-L is phosphorylated at TCR microclusters in an actin polymerization-dependent fashion. Furthermore, Cas-L participates in a positive feedback loop leading to amplification of Ca2+ signaling, inside–out integrin activation, and actomyosin contraction. We propose a new role for Cas-L in T-cell activation as a mechanical transducer linking TCR microclusters to the underlying actin network and coordinating multiple actin-dependent structures in the immunological synapse. Our studies highlight the importance of mechanotransduction processes in T-cell-mediated immune responses. PMID:27359298

  6. Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems.

    PubMed

    Bialek, Julia K; Dunay, Gábor A; Voges, Maike; Schäfer, Carola; Spohn, Michael; Stucka, Rolf; Hauber, Joachim; Lange, Ulrike C

    2016-01-01

    CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs), act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5' long terminal repeat (LTR), for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination.

  7. Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems

    PubMed Central

    Bialek, Julia K.; Dunay, Gábor A.; Voges, Maike; Schäfer, Carola; Spohn, Michael; Stucka, Rolf; Hauber, Joachim; Lange, Ulrike C.

    2016-01-01

    CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs), act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5’ long terminal repeat (LTR), for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination. PMID:27341108

  8. Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System.

    PubMed

    Qazi, Shefah; Miettinen, Heini M; Wilkinson, Royce A; McCoy, Kimberly; Douglas, Trevor; Wiedenheft, Blake

    2016-03-07

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA-guided endonucleases are powerful new tools for targeted genome engineering. These nucleases provide an efficient and precise method for manipulating eukaryotic genomes; however, delivery of these reagents to specific cell-types remains challenging. Virus-like particles (VLPs) derived from bacteriophage P22, are robust supramolecular protein cage structures with demonstrated utility for cell type-specific delivery of encapsulated cargos. Here, we genetically fuse Cas9 to a truncated form of the P22 scaffold protein, which acts as a template for capsid assembly as well as a specific encapsulation signal for Cas9. Our results indicate that Cas9 and a single-guide RNA are packaged inside the P22 VLP, and activity assays indicate that this RNA-guided endonuclease is functional for sequence-specific cleavage of dsDNA targets. This work demonstrates the potential for developing P22 as a delivery vehicle for cell specific targeting of Cas9.

  9. p130Cas alters the differentiation potential of mammary luminal progenitors by deregulating c-Kit activity.

    PubMed

    Tornillo, Giusy; Elia, Angela Rita; Castellano, Isabella; Spadaro, Michela; Bernabei, Paola; Bisaro, Brigitte; Camacho-Leal, Maria Del Pilar; Pincini, Alessandra; Provero, Paolo; Sapino, Anna; Turco, Emilia; Defilippi, Paola; Cabodi, Sara

    2013-07-01

    It has recently been proposed that defective differentiation of mammary luminal progenitors predisposes to basal-like breast cancer. However, the molecular and cellular mechanisms involved are still unclear. Here, we describe that the adaptor protein p130Cas is a crucial regulator of mouse mammary epithelial cell (MMEC) differentiation. Using a transgenic mouse model, we show that forced p130Cas overexpression in the luminal progenitor cell compartment results in the expansion of luminal cells, which aberrantly display basal cell features and reduced differentiation in response to lactogenic stimuli. Interestingly, MMECs overexpressing p130Cas exhibit hyperactivation of the tyrosine kinase receptor c-Kit. In addition, we demonstrate that the constitutive c-Kit activation alone mimics p130Cas overexpression, whereas c-Kit downregulation is sufficient to re-establish proper differentiation of p130Cas overexpressing cells. Overall, our data indicate that high levels of p130Cas, via abnormal c-Kit activation, promote mammary luminal cell plasticity, thus providing the conditions for the development of basal-like breast cancer. Consistently, p130Cas is overexpressed in human triple-negative breast cancer, further suggesting that p130Cas upregulation may be a priming event for the onset of basal-like breast cancer.

  10. Nucleosomes Selectively Inhibit Cas9 Off-target Activity at a Site Located at the Nucleosome Edge.

    PubMed

    Hinz, John M; Laughery, Marian F; Wyrick, John J

    2016-11-25

    Nucleosomes affect Cas9 binding and activity at on-target sites, but their impact at off-target sites is unknown. To investigate how nucleosomes affect Cas9 cleavage at off-target sites in vitro, we used a single guide RNA (sgRNA) that has been previously shown to efficiently direct Cas9 cleavage at the edge of the strongly positioned 601 nucleosome. Our data indicate that single mismatches between the sgRNA and DNA target have relatively little effect on Cas9 cleavage of naked DNA substrates, but strongly inhibit cleavage of nucleosome substrates, particularly when the mismatch is in the sgRNA "seed" region. These findings indicate that nucleosomes may enhance Cas9 specificity by inhibiting cleavage of off-target sites at the nucleosome edge.

  11. Photometric and Polarimetric Activity of the Herbig Ae Star VX Cas

    NASA Astrophysics Data System (ADS)

    Shakhovskoi, D. N.; Rostopchina, A. N.; Grinin, V. P.; Minikulov, N. Kh.

    2003-04-01

    We present the results of our simultaneous photometric and polarimetric observations of the Herbig Ae/Be star VX Cas acquired in 1987 2001. The star belongs to the UX Ori subtype of young variable stars and exhibits a rather low level of photometric activity: only six Algol-like minima with amplitudes ΔV>1m were recorded in 15 years of observations. Two of these minima, in 1998 and 2001, were the deepest in the history of the star’s photometric studies, with V amplitudes of about 2m. In each case, the dimming was accompanied by an increase in the linear polarization in agreement with the law expected for variable circumstellar extinction. The highest V polarization was about 5%. Observations of VX Cas in the deep minima revealed a turnover of the color tracks, typical of stars of this type and due to an increased contribution from radiation scattered in the circumstellar disk. We separated the observed polarization of VX Cas into interstellar (P is) and intrinsic (P in) components. Their position angles differ by approximately 60°, with P is dominating in the bright state and P in dominating during the deep minima. The competition of these two polarization components leads to changes in both the degree and position angle of the polarization during the star’s brightness variations. Generally speaking, in terms of the behavior of the brightness, color indices, and linear polarization, VX Cas is similar to other UX Ori stars studied by us earlier. A number of episodes of photometric and polarimetric activity suggest that, in their motion along highly eccentric orbits, circumstellar gas and dust clouds can enter the close vicinity of the star (and be disrupted there).

  12. Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity.

    PubMed

    Nuñez, James K; Kranzusch, Philip J; Noeske, Jonas; Wright, Addison V; Davies, Christopher W; Doudna, Jennifer A

    2014-06-01

    The initial stage of CRISPR-Cas immunity involves the integration of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved among all CRISPR-Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-Å-resolution crystal structure of the Cas1-Cas2 complex. Mutations that perturb Cas1-Cas2 complex formation disrupt CRISPR DNA recognition and spacer acquisition in vivo. Active site mutants of Cas2, unlike those of Cas1, can still acquire new spacers, thus indicating a nonenzymatic role of Cas2 during immunity. These results reveal the universal roles of Cas1 and Cas2 and suggest a mechanism by which Cas1-Cas2 complexes specify sites of CRISPR spacer integration.

  13. Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.

    PubMed

    Nam, Ki Hyun; Ding, Fran; Haitjema, Charles; Huang, Qingqiu; DeLisa, Matthew P; Ke, Ailong

    2012-10-19

    The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5'-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg(2+) or Mn(2+)), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1-α1 loop.

  14. Membrane bioreactor (MBR) sludge inoculation in a hybrid process scheme concept to assist overloaded conventional activated sludge (CAS) process operations.

    PubMed

    Fenu, A; Roels, J; Van Damme, S; Wambecq, T; Weemaes, M; Thoeye, C; De Gueldre, G; Van De Steene, B

    2012-01-01

    This study analyzes the effect of inoculating membrane bioreactor (MBR) sludge in a parallel-operated overloaded conventional activated sludge (CAS) system. Modelling studies that showed the beneficial effect of this inoculation were confirmed though full scale tests. Total nitrogen (TN) removal in the CAS increased and higher nitrate formation rates were achieved. During MBR sludge inoculation, the TN removal in the CAS was proven to be dependent on MBR sludge loading. Special attention was given to the effect of inoculation on sludge quality. The MBR flocs, grown without selection pressure, were clearly distinct from the more compact flocs in the CAS system and also contained more filamentous bacteria. After inoculation the MBR flocs did not evolve into good-settling compact flocs, resulting in a decreasing sludge quality. During high flow conditions the effluent CAS contained more suspended solids. Sludge volume index, however, did not increase. Laboratory tests were held to determine the threshold volume of MBR sludge to be seeded into the CAS reactor. Above 16-30%, supernatant turbidity and scum formation increased markedly.

  15. Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived transcriptional activators.

    PubMed

    Bogerd, Hal P; Kornepati, Anand V R; Marshall, Joy B; Kennedy, Edward M; Cullen, Bryan R

    2015-12-29

    Whereas several mammalian proteins can restrict the replication of HIV-1 and other viruses, these are often not expressed in relevant target cells. A potential method to inhibit viral replication might therefore be to use synthetic transcription factors to induce restriction factor expression. In particular, mutants of the RNA-guided DNA binding protein Cas9 that have lost their DNA cleavage activity could be used to recruit transcription activation domains to specific promoters. However, initial experiments revealed only weak activation unless multiple promoter-specific single guide RNAs (sgRNAs) were used. Recently, the recruitment of multiple transcription activation domains by a single sgRNA, modified to contain MS2-derived stem loops that recruit fusion proteins consisting of the MS2 coat protein linked to transcription activation domains, was reported to induce otherwise silent cellular genes. Here, we demonstrate that such "synergistic activation mediators" can induce the expression of two restriction factors, APOBEC3G (A3G) and APOBEC3B (A3B), in human cells that normally lack these proteins. We observed modest activation of endogenous A3G or A3B expression using single sgRNAs but high expression when two sgRNAs were used. Whereas the induced A3G and A3B proteins both blocked infection by an HIV-1 variant lacking a functional vif gene by inducing extensive dC-to-dU editing, only the induced A3B protein inhibited wild-type HIV-1. These data demonstrate that Cas9-derived transcriptional activators have the potential to be used for screens for endogenous genes that affect virus replication and raise the possibility that synthetic transcription factors might prove clinically useful if efficient delivery mechanisms could be developed.

  16. Canadian space robotic activities

    NASA Astrophysics Data System (ADS)

    Sallaberger, Christian; Space Plan Task Force, Canadian Space Agency

    The Canadian Space Agency has chosen space robotics as one of its key niche areas, and is currently preparing to deliver the first flight elements for the main robotic system of the international space station. The Mobile Servicing System (MSS) is the Canadian contribution to the international space station. It consists of three main elements. The Space Station Remote Manipulator System (SSRMS) is a 7-metre, 7-dof, robotic arm. The Special Purpose Dextrous Manipulator (SPDM), a smaller 2-metre, 7-dof, robotic arm can be used independently, or attached to the end of the SSRMS. The Mobile Base System (MBS) will be used as a support platform and will also provide power and data links for both the SSRMS and the SPDM. A Space Vision System (SVS) has been tested on Shuttle flights, and is being further developed to enhance the autonomous capabilities of the MSS. The CSA also has a Strategic Technologies in Automation and Robotics Program which is developing new technologies to fulfill future robotic space mission needs. This program is currently developing in industry technological capabilities in the areas of automation of operations, autonomous robotics, vision systems, trajectory planning and object avoidance, tactile and proximity sensors, and ground control of space robots. Within the CSA, a robotic testbed and several research programs are also advancing technologies such as haptic devices, control via head-mounted displays, predictive and preview displays, and the dynamic characterization of robotic arms. Canada is also now developing its next Long Term Space Plan. In this context, a planetary exploration program is being considered, which would utilize Canadian space robotic technologies in this new arena.

  17. Fluctuation of the CaS -sequestering activity of permeabilized sea urchin embryos during the cell cycle

    SciTech Connect

    Suprynowicz, F.A.; Mazia, D.

    1985-04-01

    The authors have followed the sequestration of CaS by intracellular compartments in sea urchin embryos through the first cell cycles. To gain biochemical access to these compartments, the embryos were permeabilized by brief exposure to an intense electric field. Sequestration was determined as the retention of tracer, UVCa, after filtration of aliquots on Millipore filters. The permeabilized cells sequester CaS at a constant rate for at least 20 min. The CaS -sequestering activities of embryos that are permeabilized at successive stages of the first cell cycle (one-cell stage) progressively increase to 5 times the initial level. The rate of sequestration is maximal during telophase and, in some populations of zygotes, is nearly as great throughout prophase. Over the course of the second cell cycle (two-cell stage), the activity undergoes a 2-fold oscillation that bears the same temporal relationship to mitosis as the previous fluctuation.

  18. Second-Order Perturbation Theory for Generalized Active Space Self-Consistent-Field Wave Functions.

    PubMed

    Ma, Dongxia; Li Manni, Giovanni; Olsen, Jeppe; Gagliardi, Laura

    2016-07-12

    A multireference second-order perturbation theory approach based on the generalized active space self-consistent-field (GASSCF) wave function is presented. Compared with the complete active space (CAS) and restricted active space (RAS) wave functions, GAS wave functions are more flexible and can employ larger active spaces and/or different truncations of the configuration interaction expansion. With GASSCF, one can explore chemical systems that are not affordable with either CASSCF or RASSCF. Perturbation theory to second order on top of GAS wave functions (GASPT2) has been implemented to recover the remaining electron correlation. The method has been benchmarked by computing the chromium dimer ground-state potential energy curve. These calculations show that GASPT2 gives results similar to CASPT2 even with a configuration interaction expansion much smaller than the corresponding CAS expansion.

  19. Cas9 Functionally Opens Chromatin

    PubMed Central

    Barkal, Amira A.; Srinivasan, Sharanya; Hashimoto, Tatsunori; Gifford, David K.; Sherwood, Richard I.

    2016-01-01

    Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci. Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs. Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding. PMID:27031353

  20. Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9.

    PubMed

    Koo, Taeyoung; Lee, Jungjoon; Kim, Jin-Soo

    2015-06-01

    Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations.

  1. An active immune defense with a minimal CRISPR (clustered regularly interspaced short palindromic repeats) RNA and without the Cas6 protein.

    PubMed

    Maier, Lisa-Katharina; Stachler, Aris-Edda; Saunders, Sita J; Backofen, Rolf; Marchfelder, Anita

    2015-02-13

    The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3' handle are still active in triggering an interference reaction. The complete 3' handle could be removed without loss of activity. However, manipulations of the 5' handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference.

  2. Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri.

    PubMed

    Sanozky-Dawes, Rosemary; Selle, Kurt; O'Flaherty, Sarah; Klaenhammer, Todd; Barrangou, Rodolphe

    2015-09-01

    Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (cas), which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbour a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically and medically important microbes. In this study, 17 Lactobacillus gasseri strains were investigated and 6 harboured a type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the protospacer adjacent motif sequence, determined to be 5'-NTAA-3' flanking the 3' end of the protospacer. The system in L. gasseri JV-V03 and NCK 1342 interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native type II-A CRISPR-Cas system in the commensal species L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri, and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria.

  3. sgRNA Scorer 2.0: A Species-Independent Model To Predict CRISPR/Cas9 Activity.

    PubMed

    Chari, Raj; Yeo, Nan Cher; Chavez, Alejandro; Church, George M

    2017-02-10

    It has been possible to create tools to predict single guide RNA (sgRNA) activity in the CRISPR/Cas9 system derived from Streptococcus pyogenes due to the large amount of data that has been generated in sgRNA library screens. However, with the discovery of additional CRISPR systems from different bacteria, which show potent activity in eukaryotic cells, the approach of generating large data sets for each of these systems to predict their activity is not tractable. Here, we present a new guide RNA tool that can predict sgRNA activity across multiple CRISPR systems. In addition to predicting activity for Cas9 from S. pyogenes and Streptococcus thermophilus CRISPR1, we experimentally demonstrate that our algorithm can predict activity for Cas9 from Staphylococcus aureus and S. thermophilus CRISPR3. We also have made available a new version of our software, sgRNA Scorer 2.0, which will allow users to identify sgRNA sites for any PAM sequence of interest.

  4. Space weather activities in Europe

    NASA Astrophysics Data System (ADS)

    Wimmer-Schweingruber, R. F.

    The Sun has long been understood as a source of energy for mankind. Only in the more modern times has it also been seen as a source of disturbances in the space environment of the Earth, but also of the other planets and the heliosphere. Space weather research had an early start in Europe with investigations of Birkeland, Fitzgerald and Lodge, ultimately leading to an understanding of geomagnetic storms and their relation to the Sun. Today, European space weather activities range from the study of the Sun, through the inner heliosphere, to the magnetosphere, ionosphere, atmosphere, down to ground level effects. We will give an overview of European space weather activities and focus on the chain of events from Sun to Earth.

  5. An Efficient Visual Screen for CRISPR/Cas9 Activity in Arabidopsis thaliana.

    PubMed

    Hahn, Florian; Mantegazza, Otho; Greiner, André; Hegemann, Peter; Eisenhut, Marion; Weber, Andreas P M

    2017-01-01

    The CRISPR/Cas9 system enables precision editing of the genome of the model plant Arabidopsis thaliana and likely of any other organism. Tools and methods for further developing and optimizing this widespread and versatile system in Arabidopsis would hence be welcomed. Here, we designed a generic vector system that can be used to clone any sgRNA sequence in a plant T-DNA vector containing an ubiquitously expressed Cas9 gene. With this vector, we explored two alternative marker systems for tracking Cas9-mediated gene-editing in vivo: BIALAPHOS RESISTANCE (BAR) and GLABROUS1 (GL1). BAR confers resistance to glufosinate and is widely used as a positive selection marker; GL1 is required for the formation of trichomes. Reversion of a frameshift null BAR allele to a functional one by Cas9-mediated gene editing yielded a higher than expected number of plants that are resistant to glufosinate. Surprisingly, many of those plants did not display reversion of the BAR gene through the germline. We hypothesize that few BAR revertant cells in a highly chimeric plant likely provide system-wide resistance to glufosinate and thus we suggest that BAR is not suitable as marker for tracking Cas9-mediated gene-editing. Targeting the GL1 gene for disruption with Cas9 provided clearly visible phenotypes of partially and completely glabrous plants. 50% of the analyzed T1 plants produced descendants with a chimeric phenotype and we could recover fully homozygous plants in the T3 generation with high efficiency. We propose that targeting of GL1 is suitable for assessing and optimizing Cas9-mediated gene-editing in Arabidopsis.

  6. An Efficient Visual Screen for CRISPR/Cas9 Activity in Arabidopsis thaliana

    PubMed Central

    Hahn, Florian; Mantegazza, Otho; Greiner, André; Hegemann, Peter; Eisenhut, Marion; Weber, Andreas P. M.

    2017-01-01

    The CRISPR/Cas9 system enables precision editing of the genome of the model plant Arabidopsis thaliana and likely of any other organism. Tools and methods for further developing and optimizing this widespread and versatile system in Arabidopsis would hence be welcomed. Here, we designed a generic vector system that can be used to clone any sgRNA sequence in a plant T-DNA vector containing an ubiquitously expressed Cas9 gene. With this vector, we explored two alternative marker systems for tracking Cas9-mediated gene-editing in vivo: BIALAPHOS RESISTANCE (BAR) and GLABROUS1 (GL1). BAR confers resistance to glufosinate and is widely used as a positive selection marker; GL1 is required for the formation of trichomes. Reversion of a frameshift null BAR allele to a functional one by Cas9-mediated gene editing yielded a higher than expected number of plants that are resistant to glufosinate. Surprisingly, many of those plants did not display reversion of the BAR gene through the germline. We hypothesize that few BAR revertant cells in a highly chimeric plant likely provide system-wide resistance to glufosinate and thus we suggest that BAR is not suitable as marker for tracking Cas9-mediated gene-editing. Targeting the GL1 gene for disruption with Cas9 provided clearly visible phenotypes of partially and completely glabrous plants. 50% of the analyzed T1 plants produced descendants with a chimeric phenotype and we could recover fully homozygous plants in the T3 generation with high efficiency. We propose that targeting of GL1 is suitable for assessing and optimizing Cas9-mediated gene-editing in Arabidopsis. PMID:28174584

  7. Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems.

    PubMed

    Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B

    2015-01-01

    Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing.

  8. US Space Situational Awareness activities

    NASA Astrophysics Data System (ADS)

    Butler, Stephen, , Col; Hand, Kelly; Smith, Bradley, , Col

    A new ESA Programme on Space Situational Awareness (SSA) has been approved during the ESA Council at Ministerial level in November 2008. A preparatory phase is in progress, covering the timeframe 2009 -2012. It concentrates on the architectural design of the SSA System, its governance and data policy, as well as on the provision of precursor services based on the federation of existing National and European assets. A continuation of the SSA programme will be proposed at the next Ministerial Council for the years 2012 and onwards. The SSA Preparatory Programme covers three distinct segments, namely: -Space Surveillance and Tracking of artificial objects orbiting the Earth -Space Weather -Near Earth Objects Each of the above segments has a strong relation with Science and is supported by specific RD Programmes at National, EC and ESA levels. In this paper, the scientific aspects of the three SSA Segments are outlined and the following main topics developed: • Space Surveillance: statistical models of the evolution of the space debris population in Earth-bound orbits, study of active mitigation measures, impact analysis, tracking and char-acterisation principles based on radar and optical techniques. • Space Weather: awareness of the natural space environment, detection and forecasting of space weather effects and interferences, analysis of appropriate ground and space-based sensors for the monitoring of the Sun, the solar wind, the radiation belts, the magnetosphere and the ionosphere. • Near Earth Objects (NEOs): methods for determination of physical characteristics of newly discovered objects, study of appropriate sensors based on radar and optical techniques, iden-tification and ranking of collision risks of NEOs with the Earth, study of possible mitigation measures (e.g. Don Quichotes project). The research topics undertaken during the preparatory programme, as well as those foreseen during the next phase, possibly with a strong international cooperation

  9. Extravehicular activity space suit interoperability

    NASA Astrophysics Data System (ADS)

    Skoog, A. Ingemar; McBarron, James W.; Severin, Guy I.

    1995-10-01

    The European Agency (ESA) and the Russian Space Agency (RKA) are jointly developing a new space suit system for improved extravehicular activity (EVA) capabilities in support of the MIR Space Station Programme, the EVA Suit 2000. Recent national policy agreements between the U.S. and Russia on planned cooperations in manned space also include joint extravehicular activity (EVA). With an increased number of space suit systems and a higher operational frequency towards the end of this century an improved interoperability for both routine and emergency operations is of eminent importance. It is thus timely to report the current status of ongoing work on international EVA interoperability being conducted by the Committee on EVA Protocols and Operations of the International Academy of Astronautics initialed in 1991. This paper summarises the current EVA interoperability issues to be harmonised and presents quantified vehicle interface requirements for the current U.S. Shuttle EMU and Russian MIR Orlan DMA and the new European/Russian EVA Suit 2000 extravehicular systems. Major critical/incompatible interfaces for suits/mothercraft of different combinations arc discussed, and recommendations for standardisations given.

  10. Fate of antibiotics in activated sludge followed by ultrafiltration (CAS-UF) and in a membrane bioreactor (MBR).

    PubMed

    Sahar, Eyal; Messalem, Rami; Cikurel, Haim; Aharoni, Avi; Brenner, Asher; Godehardt, Manuel; Jekel, Martin; Ernst, Mathias

    2011-10-15

    The fates of several macrolide, sulphonamide, and trimethoprim antibiotics contained in the raw sewage of the Tel-Aviv wastewater treatment plant (WWTP) were investigated after the sewage was treated using either a full-scale conventional activated sludge (CAS) system coupled with a subsequent ultrafiltration (UF) step or a pilot membrane bioreactor (MBR) system. Antibiotics removal in the MBR system, once it achieved stable operation, was 15-42% higher than that of the CAS system. This advantage was reduced to a maximum of 20% when a UF was added to the CAS. It was hypothesized that the contribution of membrane separation (in both systems) to antibiotics removal was due either to sorption to biomass (rather than improvement in biodegradation) or to enmeshment in the membrane biofilm (since UF membrane pores are significantly larger than the contaminant molecules). Batch experiments with MBR biomass showed a markedly high potential for sorption of the tested antibiotics onto the biomass. Moreover, methanol extraction of MBR biomass released significant amounts of sorbed antibiotics. This finding implies that more attention must be devoted to the management of excess sludge.

  11. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

    PubMed

    Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

    2014-04-01

    The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

  12. Active probing of space plasmas

    NASA Astrophysics Data System (ADS)

    Chan, Chang; Silevitch, Michael B.; Villalon, Elena

    1989-09-01

    During the course of the research period our efforts were focused on the following areas: (1) An examination of stochastic acceleration mechanisms in the ionosphere; (2) A study of nonequilibrium dynamics of the coupled magnetosphere - ionosphere system; and (3) Laboratory studies of active space experiments. Reprints include: Dynamics of charged particles in the near wake of a very negatively charged body -- Laboratory experiment and numerical simulation; Laboratory study of the electron temperature in the near wake of a conducting body; New model for auroral breakup during substorms; Substorm breakup on closed field lines; New model for substorm on sets -- The pre-breakup and triggering regimes; Model of the westward traveling surge and the generation of Pi 2 pulsations; Ionospheric electron acceleration by electromagnetic waves near regions of plasma resonances; Relativistic particle acceleration by obliquely propagating electromagnetic fields; Some consequences of intense electromagnetic wave injection into space plasmas.

  13. Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

    PubMed Central

    Elmore, Joshua R.; Sheppard, Nolan F.; Ramia, Nancy; Deighan, Trace; Li, Hong; Terns, Rebecca M.; Terns, Michael P.

    2016-01-01

    CRISPR–Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM. PMID:26848045

  14. Photospheric Activity in Selected Be STARS: lambda Eri and gamma Cas

    NASA Technical Reports Server (NTRS)

    Smith, Myron A.

    1994-01-01

    Recent observations of rapid variations in optical He I lines, X-rays, and FUV wavelengths in the prototypical classical Be stars lambda Eri and star gamma Cas hint that the violent processes occur on the surfaces of these stars almost all the time. We suggest that of these phenomena show greater similarities with magnetic flaring than any other process through to occur on stars.

  15. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9

    PubMed Central

    Donovan, Katherine F.; Smith, Ian; Tothova, Zuzana; Wilen, Craig; Orchard, Robert; Virgin, Herbert W.; Root, David E.

    2015-01-01

    CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), Cas9 can be reprogrammed to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently-devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering. PMID:26780180

  16. Trends in space activities in 2014: The significance of the space activities of governments

    NASA Astrophysics Data System (ADS)

    Paikowsky, Deganit; Baram, Gil; Ben-Israel, Isaac

    2016-01-01

    This article addresses the principal events of 2014 in the field of space activities, and extrapolates from them the primary trends that can be identified in governmental space activities. In 2014, global space activities centered on two vectors. The first was geopolitical, and the second relates to the matrix between increasing commercial space activities and traditional governmental space activities. In light of these two vectors, the article outlines and analyzes trends of space exploration, human spaceflights, industry and technology, cooperation versus self-reliance, and space security and sustainability. It also reviews the space activities of the leading space-faring nations.

  17. A Census of the Class of X-ray Active γ Cas Stars

    NASA Astrophysics Data System (ADS)

    Smith, M. A.; de Oliveira, R. L.; Motch, C.

    2016-11-01

    “γ Cas stars” are perhaps the primary contributors to the total hard X-ray flux from Galactic B stars. We review basic properties of 12 suspected or known members of this class. The sample extends out to 6-7 kpc and is finally sufficient to compare such basic properties as spectral type, rotation rate, binarity/Blue Straggler status, Hα lobe structure EWHα, Lx, temperature of the dominant X-ray emitting plasma component (kThot), and ranges in Lx and kThot.

  18. Space Research, Education, and Related Activities in the Space Sciences

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The Universities Space Research Association received an award of Cooperative Agreement #NCC5-356 on September 29, 1998. The mission of this activity, know as the Cooperative Program in Space Sciences (CPSS), is to conduct space science research and leading-edge instrumentation and technology development, enable research by the space sciences communities, and to expedite the effective dissemination of space science research, technology, data, and information to the educational community and the general public. To fulfill this mission, USRA recruits and maintains a staff of scientific researchers, operates a series of guest investigator facilities, organizes scientific meetings and workshops, and encourages various interactions with students and university faculty members.

  19. Space Research, Education, and Related Activities in the Space Sciences

    NASA Technical Reports Server (NTRS)

    Black, David; Marshall, Frank (Technical Monitor)

    2002-01-01

    The Universities Space Research Association received an award of Cooperative Agreement NCC5-356 on September 29, 1998. The mission of this activity, known as the Cooperative Program in Space Sciences (CPSS), is to conduct space science research and leading-edge instrumentation and technology development, enable research by the space sciences communities, and to expedite the effective dissemination of space science research, technology, data, and information to the educational community and the general public. To fulfill this mission, USRA recruits and maintains a staff of scientific researchers, operates a series of guest investigator facilities, organizes scientific meetings and workshops, and encourages various interactions with students and university faculty members.

  20. Space Research, Education, and Related Activities In the Space Sciences

    NASA Technical Reports Server (NTRS)

    Black, David

    2002-01-01

    The mission of this activity, known as the Cooperative Program in Space Sciences (CPSS), is to conduct space science research and leading-edge instrumentation and technology development, enable research by the space sciences communities, and to expedite the effective dissemination of space science research, technology, data, and information to the educational community and the general public. To fulfill this mission, the Universities Space Research Association (USRA) recruits and maintains a staff of scientific researchers, operates a series of guest investigator facilities, organizes scientific meetings and workshops, and encourages various interactions with students and university faculty members. This paper is the final report from this now completed Cooperative Agreement.

  1. CasExpress reveals widespread and diverse patterns of cell survival of caspase-3 activation during development in vivo

    PubMed Central

    Ding, Austin Xun; Sun, Gongping; Argaw, Yewubdar G; Wong, Jessica O; Easwaran, Sreesankar; Montell, Denise J

    2016-01-01

    Caspase-3 carries out the executioner phase of apoptosis, however under special circumstances, cells can survive its activity. To document systematically where and when cells survive caspase-3 activation in vivo, we designed a system, CasExpress, which drives fluorescent protein expression, transiently or permanently, in cells that survive caspase-3 activation in Drosophila. We discovered widespread survival of caspase-3 activity. Distinct spatial and temporal patterns emerged in different tissues. Some cells activated caspase-3 during their normal development in every cell and in every animal without evidence of apoptosis. In other tissues, such as the brain, expression was sporadic both temporally and spatially and overlapped with periods of apoptosis. In adults, reporter expression was evident in a large fraction of cells in most tissues of every animal; however the precise patterns varied. Inhibition of caspase activity in wing discs reduced wing size demonstrating functional significance. The implications of these patterns are discussed. DOI: http://dx.doi.org/10.7554/eLife.10936.001 PMID:27058168

  2. Space activities and global popular music culture

    NASA Astrophysics Data System (ADS)

    Wessels, Allison Rae; Collins, Patrick

    During the "space age" era, space activities appear increasingly as a theme in Western popular music, as they do in popular culture generally. In combination with the electronics and tele-communications revolution, "pop/rock" music has grown explosively during the space age to become an effectively global culture. From this base a number of trends are emerging in the pattern of influences that space activities have on pop music. The paper looks at the use of themes and imagery in pop music; the role of space technology in the modern "globalization" of pop music; and current and future links between space activities and pop music culture, including how public space programmes are affected by its influence on popular attitudes.

  3. Space weather activities at NOAA s Space Environment Center

    NASA Astrophysics Data System (ADS)

    Kunches, J.

    The NOAA Space Environment Center is the focal point for real-time space weather monitoring and prediction in the United States . The Space Weather Operations (SWO) division staffs a 24-hour/day operations center, through which both in-situ and remotely sensed data and imagery flow. These diverse data streams are analyzed continuously, and that information is applied to both predictions and specifications of various aspects of the space environment. These include the behavior of the geomagnetic field, the character of the ionosphere, and the strength of the near-earth radiation environment. Models are brought to bear in each of thes e areas, as SEC has an active research-to-operations transition effort. The Rapid Prototyping Center is the venue through which pertinent models and data must pass to be brought into the operational arena. The model outputs are then made available both internally and externally. SEC is a member of the International Space Environment Service (ISES), a partnership currently consisting of eleven nations. The mission of the ISES is to encourage and facilitate near-real-time international monitoring and prediction of the space environment by: the rapid exchange of space environment information; the standardization of the methodology for space environment observations and data reduction; the uniform publication of observations and statistics; and the application of standardized space environment products and services to assist users in reducing the impact of space weather on activities of human interest. An overview of the operational attributes of the SEC, and the function of the ISES, will be presented. Additional issues related to space weather customers, new data streams to be available in the near-term, and how these new data and imagery will be integrated int o operations will be discussed.

  4. Aeronautics and space report of the President: 1981 activities

    NASA Technical Reports Server (NTRS)

    1981-01-01

    Achievements in the aeronautics and space program by function are summarized. Activities in communications, Earth's resources and environment, space science, space transportation, international activities, and aeronautics are included.

  5. Space activities in 2009/2010

    NASA Astrophysics Data System (ADS)

    Pagkratis, Spyros

    2011-09-01

    The global financial crisis of 2008 has created an economic environment unfavourable to public and corporate economic activity alike, which could not have left space activities unaffected. However, the effects of the crisis upon the space sector have been so far less damaging than anticipated. The following paper presents recent developments in the field of space policies, institutional budgets and commercial activity worldwide, in an effort to improve the understanding of the new trends in commercial and public space activities. It particularly explores the strategies followed by space stakeholders in different countries and regions in order to pursue their planned space programmes in view of difficult financial conditions. Finally, it highlights the differences in the outlook of space activities between established and emerging space-faring nations and attempts to explore their medium-term consequences on an international level. For this purpose, it was based on research conducted in the framework of a recent ESPI report on "Space Policies, Issues and trends in 2009/2010".

  6. Operational Space Weather Activities in the US

    NASA Astrophysics Data System (ADS)

    Berger, Thomas; Singer, Howard; Onsager, Terrance; Viereck, Rodney; Murtagh, William; Rutledge, Robert

    2016-07-01

    We review the current activities in the civil operational space weather forecasting enterprise of the United States. The NOAA/Space Weather Prediction Center is the nation's official source of space weather watches, warnings, and alerts, working with partners in the Air Force as well as international operational forecast services to provide predictions, data, and products on a large variety of space weather phenomena and impacts. In October 2015, the White House Office of Science and Technology Policy released the National Space Weather Strategy (NSWS) and associated Space Weather Action Plan (SWAP) that define how the nation will better forecast, mitigate, and respond to an extreme space weather event. The SWAP defines actions involving multiple federal agencies and mandates coordination and collaboration with academia, the private sector, and international bodies to, among other things, develop and sustain an operational space weather observing system; develop and deploy new models of space weather impacts to critical infrastructure systems; define new mechanisms for the transition of research models to operations and to ensure that the research community is supported for, and has access to, operational model upgrade paths; and to enhance fundamental understanding of space weather through support of research models and observations. The SWAP will guide significant aspects of space weather operational and research activities for the next decade, with opportunities to revisit the strategy in the coming years through the auspices of the National Science and Technology Council.

  7. Physics of Space Plasma Activity

    NASA Astrophysics Data System (ADS)

    Schindler, Karl

    2010-04-01

    Preface; 1. Introduction; Part I. Setting the Scene: 2. Sites of activity; 3. Plasma models; Part II. Quiescence: 4. Introduction; 5. Magnetohydrodynamic states; 6. Particle picture of steady states; 7. A unified theory of steady states; 8. Quasi-static evolution and thin current sheets (TCS); Part III. Dynamics: 9. Nonideal effects; 10. Selected macroinstabilities; 11. Magnetic reconnection; 12. Aspects of bifurcation and nonlinear dynamics; Part IV. Applications: 13. Magnetospheric activity; 14. Models of solar activity; 15. Discussion; Appendix 1. Unified theory: details and derivations; Appendix 2. Variational principle for collisionless plasmas; Appendix 3. Symbols and fundamental constants; References; Index.

  8. Activities in Science Related to Space.

    ERIC Educational Resources Information Center

    National Aeronautics and Space Administration, Washington, DC. Educational Programs Div.

    Contained are a collection of science activities based upon forty-six scientific concepts related to space science. These activities are designed for junior high school science, but a much wider grade level range of use is possible. The booklet is primarily intended for teacher use. Each series of concept-oriented activities is independent of the…

  9. Active Control of Cryogenic Propellants in Space

    NASA Technical Reports Server (NTRS)

    Notardonato, William

    2011-01-01

    A new era of space exploration is being planned. Exploration architectures under consideration require the long term storage of cryogenic propellants in space. This requires development of active control systems to mitigate the effect of heat leak. This work summarizes current state of the art, proposes operational design strategies and presents options for future architectures. Scaling and integration of active systems will be estimated. Ideal long range spacecraft systems will be proposed with Exploration architecture benefits considered.

  10. Landsat: Space Activities for Students

    ERIC Educational Resources Information Center

    Marks, Steven K.

    1979-01-01

    An aerospace education activity is described which is suitable for grades 3-12. Students piece together several images from the Landsat satellite to make a mosaic of their state. From the mosaic clear acetate overlay maps can be made relating to such subjects as agriculture, geology, hydrology, or urban planning. (BB)

  11. Genome modification by CRISPR/Cas9.

    PubMed

    Ma, Yuanwu; Zhang, Lianfeng; Huang, Xingxu

    2014-12-01

    Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)9-mediated genome modification enables us to edit the genomes of a variety of organisms rapidly and efficiently. The advantages of the CRISPR-Cas9 system have made it an increasingly popular genetic engineering tool for biological and therapeutic applications. Moreover, CRISPR-Cas9 has been employed to recruit functional domains that repress/activate gene expression or label specific genomic loci in living cells or organisms, in order to explore developmental mechanisms, gene expression regulation, and animal behavior. One major concern about this system is its specificity; although CRISPR-Cas9-mediated off-target mutation has been broadly studied, more efforts are required to further improve the specificity of CRISPR-Cas9. We will also discuss the potential applications of CRISPR-Cas9.

  12. Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine.

    PubMed

    Gersbach, Charles A; Perez-Pinera, Pablo

    2014-08-01

    New technologies have recently been developed to control the expression of human genes in their native genomic context by engineering synthetic transcription factors that can be targeted to any DNA sequence. The ability to precisely regulate any gene as it occurs naturally in the genome provides a means to address a variety of diseases and disorders. This approach also circumvents some of the traditional challenges of gene therapy. In this editorial, we review the technologies that have enabled targeted human gene activation, including the engineering of transcription factors based on zinc finger proteins, transcription activator-like effectors and the CRISPR/Cas9 system. Additionally, we highlight examples in which these methods have been developed for therapeutic applications and discuss challenges and opportunities.

  13. High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system.

    PubMed

    Yosef, Ido; Goren, Moran G; Kiro, Ruth; Edgar, Rotem; Qimron, Udi

    2011-12-13

    Prokaryotic DNA arrays arranged as clustered regularly interspaced short palindromic repeats (CRISPR), along with their associated proteins, provide prokaryotes with adaptive immunity by RNA-mediated targeting of alien DNA or RNA matching the sequences between the repeats. Here, we present a thorough screening system for the identification of bacterial proteins participating in immunity conferred by the Escherichia coli CRISPR system. We describe the identification of one such protein, high-temperature protein G (HtpG), a homolog of the eukaryotic chaperone heat-shock protein 90. We demonstrate that in the absence of htpG, the E. coli CRISPR system loses its suicidal activity against λ prophage and its ability to provide immunity from lysogenization. Transcomplementation of htpG restores CRISPR activity. We further show that inactivity of the CRISPR system attributable to htpG deficiency can be suppressed by expression of Cas3, a protein that is essential for its activity. Accordingly, we also find that the steady-state level of overexpressed Cas3 is significantly enhanced following HtpG expression. We conclude that HtpG is a newly identified positive modulator of the CRISPR system that is essential for maintaining functional levels of Cas3.

  14. Movement Activities for Places and Spaces.

    ERIC Educational Resources Information Center

    Rasmus, Carolyn J., Ed.; Fowler, John, Ed.

    This manual is for the use of elementary school teachers. It presents a systematic approach to teaching movement and ways of teaching physical education activities in the classroom rather than in a gymnasium or out-of-doors. Activity games that will help children develop flexibility, motor skills, and a sense of space and cooperation with others…

  15. Space based astronomy: Teacher's guide with activities

    NASA Technical Reports Server (NTRS)

    Rosenberg, Carla B. (Editor); Weiler, Edward; Morrow, Cherilyn; Bacon, Pamela M.; Thorne, Muriel; Blanchard, Paul A.; Howard, Sethane; Pengra, Patricia R.; Brown, Deborah A.; Winrich, Ralph

    1994-01-01

    This curriculum guide uses hands-on activities to help students and teachers understand the significance of space-based astronomy - astronomical observations made from outer space. The guide contains few of the traditional activities found in many astronomy guides such as constellation studies, lunar phases, and planetary orbits. Instead, it tells the story of why it is important to observe celestial objects from outer space and how to study the entire electromagnetic spectrum. The guide begins with a survey of astronomy related NASA spacecraft. This is followed by a collection of activities in four units: (1) the atmospheric filter; (2) the electromagnetic spectrum; (3) collecting electromagnetic radiation; and (4) down to Earth. A curriculum index identifies the curriculum areas each activity addresses. The guide concludes with a glossary, reference list, a NASA Resources list, and an evaluation card. It is designed for students in grades 5 through 8.

  16. Activities of NICT space weather project

    NASA Astrophysics Data System (ADS)

    Murata, Ken T.; Nagatsuma, Tsutomu; Watari, Shinichi; Shinagawa, Hiroyuki; Ishii, Mamoru

    NICT (National Institute of Information and Communications Technology) has been in charge of space weather forecast service in Japan for more than 20 years. The main target region of the space weather is the geo-space in the vicinity of the Earth where human activities are dominant. In the geo-space, serious damages of satellites, international space stations and astronauts take place caused by energetic particles or electromagnetic disturbances: the origin of the causes is dynamically changing of solar activities. Positioning systems via GPS satellites are also im-portant recently. Since the most significant effect of positioning error comes from disturbances of the ionosphere, it is crucial to estimate time-dependent modulation of the electron density profiles in the ionosphere. NICT is one of the 13 members of the ISES (International Space Environment Service), which is an international assembly of space weather forecast centers under the UNESCO. With help of geo-space environment data exchanging among the member nations, NICT operates daily space weather forecast service every day to provide informa-tion on forecasts of solar flare, geomagnetic disturbances, solar proton event, and radio-wave propagation conditions in the ionosphere. The space weather forecast at NICT is conducted based on the three methodologies: observations, simulations and informatics (OSI model). For real-time or quasi real-time reporting of space weather, we conduct our original observations: Hiraiso solar observatory to monitor the solar activity (solar flare, coronal mass ejection, and so on), domestic ionosonde network, magnetometer HF radar observations in far-east Siberia, and south-east Asia low-latitude ionosonde network (SEALION). Real-time observation data to monitor solar and solar-wind activities are obtained through antennae at NICT from ACE and STEREO satellites. We have a middle-class super-computer (NEC SX-8R) to maintain real-time computer simulations for solar and solar

  17. Activities of the Space Studies Board

    NASA Technical Reports Server (NTRS)

    1994-01-01

    This 1993 annual report of the Space Studies Board of the National research Council (NRC) describes the activities of the Board during a year filled with questions and change in the nation's civil space program. The accounts contained in this report briefly describe the activities of the Board and its committees and sketch out major space research issues. Two major reports are summarized, and the full text of three letter reports is included. Items considered include: (1) robotic missions to explore the Earth, the solar system, and the far reaches of the universe; (2) instability in the human flight program; (3) the redesign of the International Space Station; and (4) federal funding of research in all fields, especially basic research.

  18. Economic benefits of commercial space activities

    NASA Technical Reports Server (NTRS)

    Stone, Barbara A.

    1988-01-01

    This paper discusses the current and potential impact on the economy of selected private sector space activities including materials processing in space and satellite communications. Spacehab, a commercially developed and manufactured pressurized metal cylinder which fits in the Shuttle payload bay and connects to the crew compartment is examined along with potential uses of the Shuttle external tank. Private sector upper stage development, the privatization of expendable launch vehicles, and the transfer of NASA technology are discussed.

  19. Economic benefits of commercial space activities

    NASA Astrophysics Data System (ADS)

    Stone, Barbara A.

    Space is not only an endless frontier for exploration, but also a potentially rich arena for profitable commerce to benefit all mankind. Access to the unique environment of space provides opportunities for unprecedented kinds of research to develop new products and services. This research can lead to commercially viable enterprises, which will become permanent businesses, which will provide good jobs for workers, pay taxes to their governments, and return dividends to their investors. Seeking superior products and processes is vital if the economy is to grow and prosper. This paper discusses the current and potential impact on the economy of selected private sector space activities.

  20. Activities of the Space Studies Board

    NASA Technical Reports Server (NTRS)

    1994-01-01

    Since its founding as the Space Science Board in 1958, the Space Studies Board has provided independent external scientific and technical advice on the nation's civil space program. This 1991 Annual Report of the SSB and its committees represents the first of its kind. The report contains a summary of the board's meetings, complete texts of letter reports, executive summaries of full reports issued during the year, and congressional testimony. It is intended to serve as a ready reference to board activities and advisory reports in 1991.

  1. Environmental Impact Assessment and Space Activities

    NASA Astrophysics Data System (ADS)

    Viikari, L.

    these developments in way or another. In addition to national EIA regulations, there are also international agreements on EIA (i.a. the Espoo Convention) which establish their own EIA systems. In international law of outer space, environmental impact assessment is, however, not a well-established tool. The UN space treaties were drafted during a time when such consideratio ns were still not among the highest ranking items on national agendas. Therefore, these instruments fail to contain provisions regarding impact assessment, and also rest of the environmental content found in them is rather modest. The nearest equivalent to any impact assessment is contained in the Outer Space Treaty Article IX, namely the requirement of prior consultations in case of planned space activity or experiment that might cause "potentially harmful interference" with space activities of other St ates Parties. There also exist some applicable provisions on national level, such as the requirement of "formal assessment" on NASA programs of "[orbital] debris generation potential and debris mitigation options" in NASA Policy for Limiting Orbital Debris Generation (Art. 1.b). Also the national legislation of some space faring countries provides at least for the supply of some kind of information assessing the possible environmental consequences of proposed space activities. For instance, the Russian Statute on Lisencing Space Operations requires that for obtaining a license for space operation in the Russian Federation, the applicant has to supply, i.a. "documents confirming the safety of space operations (including ecological, fire and explosion safety) and the reliability of space equipment'"(Art.5.h). However, such provisions are obviously not enough for ensuring effective international regulation of the issue. The goal of this paper is to consider the usefulness of international environmental impact assessment for space activities. The space environment, however, is a unique arena in many ways

  2. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

    PubMed

    Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

    2014-05-06

    In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

  3. Near-space airships against terrorist activities

    NASA Astrophysics Data System (ADS)

    Kesenek, Ceylan

    2014-06-01

    Near-space is a region surrounding the earth which is too dense for a satellite to fly and also too thin for air breathing vehicles to fly. The near-space region which is located between 65,000 and 325,000 feet is really underutilized despite its unique potential. Near-Space airships can be used to exploit the potential of near space. Such a system can supply not only a great deal of information using ISR (Intelligence Surveillance Reconnaissance) sensors on board but also serve as a communication/data relay. Airships used in near space can cover a very wide footprint area for surveillance missions. Free of orbital mechanics these near-space assets can continue its mission for long period of time with a persistence of days and months. These assets can provide persistent intelligence for fight against terrorist activities. Terrorism is a non-state threat and doesn't have a static hierarchical structure. To fight against such an adversary an overwhelming intelligence activity must be applied. Therefore, intelligence collection and surveillance missions play a vital role in counter terrorism. Terrorists use asymmetric means of threat that require information superiority. In this study exploitation of near space by airships is analyzed for fight against terrorism. Near-space airships are analyzed according to the operational effectiveness, logistic structure and cost. Advantages and disadvantages of airships are argued in comparison with satellites and airplanes. As a result, by bridging the gap between the air and space, nearspace airships are considered to be the most important asset of warfighter especially with its operational effectiveness.

  4. CAS77 and CAS7276: A Review.

    ERIC Educational Resources Information Center

    Harrison, Isom, Jr.

    This paper describes the content, organization, specifications, and methods of use of the CAS77 and CAS7276 online files of worldwide chemical literature, databases produced by Chemical Abstracts Service and available from System Development Corporation (SDC). The scope of the databases, their unit record, their data elements, their modes of…

  5. Dissociative recombination of HCO/sup +/: Complete active space (CAS) SCF electronic structure calculations

    SciTech Connect

    Kraemer, W.P.; Hazi, A.U.

    1988-06-17

    Laboratory measurements of the dissociative recombination of electrons with HCO/sup /plus// ions were performed using the stationary microwave afterglow technique as well as in a flowing afterglow Langmuir probe (FALP) experiment. CASSCF calculations suggest that the recombination of vibrationally cold HCO/sup /plus// ions with low-energy electrons can only proceed via an indirect reaction mechanism. Three different dissociation channels are in principle available to stabilize the intermediate states formed by electron capture. Dissociation can occur along the repulsive potentials of the X /sup 2/..sigma../sup /plus// and of the first excited /sup 2/..sigma../sup /plus// and /sup 2//Pi/ states of HCO. Different electronic states of CO are produced in the three different dissociation channels and their exothermicities vary from ..delta..E/sub e/ = 7.1 eV for CO (X /sup 1/..sigma../sup /plus//) to ..delta..E/sub e/ = 1.1 eV for CO (a /sup 3//Pi/) and finally to ..delta..E/sub e/ = 0.2 eV for CO (a' /sup 3/..sigma../sup /plus//). 11 refs., 3 figs.

  6. Space activity and programs at Sofradir

    NASA Astrophysics Data System (ADS)

    Bouakka-Manesse, A.; Jamin, N.; Delannoy, A.; Fièque, B.; Leroy, C.; Pidancier, P.; Vial, L.; Chorier, P.; Péré Laperne, N.

    2016-10-01

    SOFRADIR is one of the leading companies involved in the development and manufacturing of infrared detectors for space applications. As a matter of fact, SOFRADIR is involved in many space programs from visible up to VLWIR spectral ranges. These programs concern operational missions for earth imagery, meteorology and also scientific missions for universe exploration. One of the last space detectors available at SOFRADIR is a visible - SWIR detector named Next Generation Panchromatic Detector (NGP) which is well adapted for hyperspectral, imagery and spectroscopy applications. In parallel of this new space detector, numerous programs are currently running for different kind of missions: meteorology (MTG), Copernicus with the Sentinel detectors series, Metop-SG system (3MI), Mars exploration (Mamiss, etc….)… In this paper, we present the last developments made for space activity and in particular the NGP detector. We will also present the space applications using this detector and show appropriateness of its use to answer space programs specifications, as for example those of Sentinel-5.

  7. In vitro enzymology of Cas9.

    PubMed

    Anders, Carolin; Jinek, Martin

    2014-01-01

    Cas9 is a bacterial RNA-guided endonuclease that uses base pairing to recognize and cleave target DNAs with complementarity to the guide RNA. The programmable sequence specificity of Cas9 has been harnessed for genome editing and gene expression control in many organisms. Here, we describe protocols for the heterologous expression and purification of recombinant Cas9 protein and for in vitro transcription of guide RNAs. We describe in vitro reconstitution of the Cas9-guide RNA ribonucleoprotein complex and its use in endonuclease activity assays. The methods outlined here enable mechanistic characterization of the RNA-guided DNA cleavage activity of Cas9 and may assist in further development of the enzyme for genetic engineering applications.

  8. Radiation protection for manned space activities

    NASA Technical Reports Server (NTRS)

    Jordan, T. M.

    1983-01-01

    The Earth's natural radiation environment poses a hazard to manned space activities directly through biological effects and indirectly through effects on materials and electronics. The following standard practices are indicated that address: (1) environment models for all radiation species including uncertainties and temporal variations; (2) upper bound and nominal quality factors for biological radiation effects that include dose, dose rate, critical organ, and linear energy transfer variations; (3) particle transport and shielding methodology including system and man modeling and uncertainty analysis; (4) mission planning that includes active dosimetry, minimizes exposure during extravehicular activities, subjects every mission to a radiation review, and specifies operational procedures for forecasting, recognizing, and dealing with large solar flaes.

  9. CRISPR/Cas9-mediated knockout of p22phox leads to loss of Nox1 and Nox4, but not Nox5 activity.

    PubMed

    Prior, Kim-Kristin; Leisegang, Matthias S; Josipovic, Ivana; Löwe, Oliver; Shah, Ajay M; Weissmann, Norbert; Schröder, Katrin; Brandes, Ralf P

    2016-10-01

    The NADPH oxidases are important transmembrane proteins producing reactive oxygen species (ROS). Within the Nox family, different modes of activation can be discriminated. Nox1-3 are dependent on different cytosolic subunits, Nox4 seems to be constitutively active and Nox5 is directly activated by calcium. With the exception of Nox5, all Nox family members are thought to depend on the small transmembrane protein p22phox. With the discovery of the CRISPR/Cas9-system, a tool to alter genomic DNA sequences has become available. So far, this method has not been widely used in the redox community. On such basis, we decided to study the requirement of p22phox in the Nox complex using CRISPR/Cas9-mediated knockout. Knockout of the gene of p22phox, CYBA, led to an ablation of activity of Nox4 and Nox1 but not of Nox5. Production of hydrogen peroxide or superoxide after knockout could be rescued with either human or rat p22phox, but not with the DUOX-maturation factors DUOXA1/A2. Furthermore, different mutations of p22phox were studied regarding the influence on Nox4-dependent H2O2 production. P22phox Q130* and Y121H affected maturation and activity of Nox4. Hence, Nox5-dependent O2(•-) production is independent of p22phox, but native p22phox is needed for maturation of Nox4 and production of H2O2.

  10. Highly efficient Cas9-mediated transcriptional programming.

    PubMed

    Chavez, Alejandro; Scheiman, Jonathan; Vora, Suhani; Pruitt, Benjamin W; Tuttle, Marcelle; P R Iyer, Eswar; Lin, Shuailiang; Kiani, Samira; Guzman, Christopher D; Wiegand, Daniel J; Ter-Ovanesyan, Dmitry; Braff, Jonathan L; Davidsohn, Noah; Housden, Benjamin E; Perrimon, Norbert; Weiss, Ron; Aach, John; Collins, James J; Church, George M

    2015-04-01

    The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).

  11. Edible Earth and Space Science Activities

    NASA Astrophysics Data System (ADS)

    Lubowich, D.; Shupla, C.

    2014-07-01

    In this workshop we describe using Earth and Space Science demonstrations with edible ingredients to increase student interest. We show how to use chocolate, candy, cookies, popcorn, bagels, pastries, Pringles, marshmallows, whipped cream, and Starburst candy for activities such as: plate tectonics, the interior structure of the Earth and Mars, radioactivity/radioactive dating of rocks and stars, formation of the planets, lunar phases, convection, comets, black holes, curvature of space, dark energy, and the expansion of the Universe. In addition to creating an experience that will help students remember specific concepts, edible activities can be used as a formative assessment, providing students with the opportunity to create something that demonstrates their understanding of the model. The students often eat the demonstrations. These demonstrations are an effective teaching tool for all ages, and can be adapted for cultural, culinary, and ethnic differences among the students.

  12. Structure and Engineering of Francisella novicida Cas9

    PubMed Central

    Hirano, Hisato; Gootenberg, Jonathan S.; Horii, Takuro; Abudayyeh, Omar O.; Kimura, Mika; Hsu, Patrick D.; Nakane, Takanori; Ishitani, Ryuichiro; Hatada, Izuho; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

    2016-01-01

    Summary The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA, and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5′-NGG-3′ PAM, and used the structural information to create a variant that can recognize the more relaxed 5′-YG-3′ PAM. Furthermore, we demonstrated that pre-assembled FnCas9 ribonucleoprotein complexes can be microinjected into mouse zygotes to edit endogenous sites with the 5′-YG-3′ PAMs, thus expanding the target space of the CRISPR-Cas9 toolbox. PMID:26875867

  13. Increasing spin-flips and decreasing cost: Perturbative corrections for external singles to the complete active space spin flip model for low-lying excited states and strong correlation

    SciTech Connect

    Mayhall, Nicholas J.; Head-Gordon, Martin

    2014-07-28

    An approximation to the spin-flip extended configuration interaction singles method is developed using a second-order perturbation theory approach. In addition to providing significant efficiency advantages, the new framework is general for an arbitrary number of spin-flips, with the current implementation being applicable for up to around 4 spin-flips. Two new methods are introduced: one which is developed using non-degenerate perturbation theory, spin-flip complete active-space (SF-CAS(S)), and a second quasidegenerate perturbation theory method, SF-CAS(S){sub 1}. These two approaches take the SF-CAS wavefunction as the reference, and then perturbatively includes the effect of single excitations. For the quasidegenerate perturbation theory method, SF-CAS(S){sub 1}, the subscripted “1” in the acronym indicates that a truncated denominator expansion is used to obtain an energy-independent down-folded Hamiltonian. We also show how this can alternatively be formulated in terms of an extended Lagrangian, by introducing an orthonormality constraint on the first-order wavefunction. Several numerical examples are provided, which demonstrate the ability of SF-CAS(S) and SF-CAS(S){sub 1} to describe bond dissociations, singlet-triplet gaps of organic molecules, and exchange coupling parameters for binuclear transition metal complexes.

  14. [CAS General Standards 2012

    ERIC Educational Resources Information Center

    Council for the Advancement of Standards in Higher Education, 2011

    2011-01-01

    The mission of the Council for the Advancement of Standards in Higher Education (CAS) is to promote the improvement of programs and services to enhance the quality of student learning and development. CAS is a consortium of professional associations who work collaboratively to develop and promulgate standards and guidelines and to encourage…

  15. Calibrated Ancillary System (CAS) user's guide, volume 1

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of the CAS. Volume 1 includes a general overview of the CAS relationships with other equipment, physical design, and hardware and software subsystems. In addition, a description of the user levels and tasks, an introduction to CAS operation, and an outline of general operating procedures are included.

  16. Calibrated Ancillary System (CAS) user's guide, volume 2

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of CAS. Volume 2 describes the central status and control (CSAC) procedures, supervisor procedures, and logging procedures.

  17. Calibrated Ancillary System (CAS) user's guide, volume 3

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of the CAS. Volume 3 describes logging and delogging procedures, real-time procedures, and error messages.

  18. Calibrated Ancillary System (CAS) user's guide, volume 8

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of CAS. Volume 8 describes procedures for invoking checkout software, file maintenance procedures, system manager procedures.

  19. Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity.

    PubMed

    Nuñez, James K; Lee, Amy S Y; Engelman, Alan; Doudna, Jennifer A

    2015-03-12

    Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1-Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1-Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1-Cas2-mediated adaptive immunity.

  20. Development of a space activity suit

    NASA Technical Reports Server (NTRS)

    Annis, J. F.; Webb, P.

    1971-01-01

    The development of a series of prototype space activity suit (SAS) assemblies is discussed. The SAS is a new type of pressure suit designed especially for extravehicular activity. It consists of a set of carefully tailored elastic fabric garments which have been engineered to supply sufficient counterpressure to the body to permit subjects to breath O2 at pressures up to 200 mm Hg without circulatory difficulty. A closed, positive pressure breathing system (PPBS) and a full bubble helmet were also developed to complete the system. The ultimate goal of the SAS is to improve the range of activity and decrease the energy cost of work associated with wearing conventional gas filled pressure suits. Results are presented from both laboratory (1 atmosphere) and altitude chamber tests with subjects wearing various SAS assemblies. In laboratory tests lasting up to three hours, the SAS was worn while subjects breathed O2 at pressures up to 170 mm Hg without developing physiological problems. The only physiological symptoms apparent were a moderate tachycardia related to breathing pressures above 130 mm Hg, and a small collection of edema fluid in the hands. Both problems were considered to be related to areas of under-pressurization by the garments. These problems, it is suggested, can ultimately be corrected by the development of new elastic fabrics and tailoring techniques. Energy cost of activity, and mobility and dexterity of subjects in the SAS, were found to be superior to those in comparable tests on subjects in full pressure suits.

  1. Space Station Active Thermal Control System modeling

    NASA Technical Reports Server (NTRS)

    Hye, Abdul; Lin, Chin H.

    1988-01-01

    The Space Station Active Thermal Control System (ATCS) has been modeled using modified SINDA/SINFLO programs to solve two-phase Thermo-fluid problems. The modifications include changes in several subroutines to incorporate implicit solution which allows larger time step as compared to that for explicit solutions. Larger time step saves computer time but involves larger computational error. Several runs were made using various time steps for the ATCS model. It has been found that for a reasonable approach, three times larger time step as compared to that used in explicit method is a good value which will reduce the computer time by approximately 50 percent and still maintain the accuracy of the output data to within 90 percent of the explicit values.

  2. Norwegian space activities 1958-2003. A historical overview

    NASA Astrophysics Data System (ADS)

    Røberg, Ole Anders; Collett, John Peter

    2004-10-01

    Contents: The early years of Norwegian geophysical and cosmic science. The first steps towards a national space research policy in Norway. A national space policy emerging between science and technology. A national programme for industralisation of space technology. Norway's long road to ESA membership. Norwegian space activities since joining ESA.

  3. International Cooperation and Competition in Civilian Space Activities.

    ERIC Educational Resources Information Center

    Congress of the U.S., Washington, DC. Office of Technology Assessment.

    This report assesses the state of international competition in civilian space activities, explores United States civilian objectives in space, and suggests alternative options for enhancing the overall U.S. position in space technologies. It also investigated past, present, and projected international cooperative arrangements for space activities…

  4. Space Industrialization: Manufacturing and Construction Activities. Part 2.

    ERIC Educational Resources Information Center

    Story, Charles H.

    1983-01-01

    Discusses how space industrialization will provide direct benefits for our nation and will transfer technology to the many diverse areas of human activity. Examples are the development of the Space Shuttle, the Space Studies Institute, and the LS Society (advocates for colonizing space). (NRJ)

  5. Europe/United States space activities

    NASA Technical Reports Server (NTRS)

    Bainum, P. M. (Editor); Von Bun, F. (Editor)

    1985-01-01

    Among the topics discussed are: the Olympus satellite program; trends in the Italian space technology; and ESA Space Station planning. Consideration is also given to cooperative international programs, including the Eurostar platform, the Tethered Satellite System, and the SPAS system; space science and applications programs; and the development of next generation space propulsion systems. Among the specific propulsion technologies discussed are: LOX/LR2 engines; the Ariane 5 solid propellant booster; and propulsion systems for earth-to-orbit vehicles.

  6. Protein engineering of Cas9 for enhanced function.

    PubMed

    Oakes, Benjamin L; Nadler, Dana C; Savage, David F

    2014-01-01

    CRISPR/Cas systems act to protect the cell from invading nucleic acids in many bacteria and archaea. The bacterial immune protein Cas9 is a component of one of these CRISPR/Cas systems and has recently been adapted as a tool for genome editing. Cas9 is easily targeted to bind and cleave a DNA sequence via a complementary RNA; this straightforward programmability has gained Cas9 rapid acceptance in the field of genetic engineering. While this technology has developed quickly, a number of challenges regarding Cas9 specificity, efficiency, fusion protein function, and spatiotemporal control within the cell remain. In this work, we develop a platform for constructing novel proteins to address these open questions. We demonstrate methods to either screen or select active Cas9 mutants and use the screening technique to isolate functional Cas9 variants with a heterologous PDZ domain inserted within the protein. As a proof of concept, these methods lay the groundwork for the future construction of diverse Cas9 proteins. Straightforward and accessible techniques for genetic editing are helping to elucidate biology in new and exciting ways; a platform to engineer new functionalities into Cas9 will help forge the next generation of genome-modifying tools.

  7. Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.

    PubMed

    Rauch, Benjamin J; Silvis, Melanie R; Hultquist, Judd F; Waters, Christopher S; McGregor, Michael J; Krogan, Nevan J; Bondy-Denomy, Joseph

    2017-01-12

    Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a countermeasure, numerous phages are known that produce proteins to block the function of class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific "anti-CRISPRs" present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9.

  8. 7. LESLIE WICKMAN, EVA (EXTRA VEHICULAR ACTIVITIES) SPECIALIST, IN SPACE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    7. LESLIE WICKMAN, EVA (EXTRA VEHICULAR ACTIVITIES) SPECIALIST, IN SPACE SUIT AFTER TESTING IN NEUTRAL BUOYANCY TANK. AVERAGE COST OF SUIT IS $1,000,000. - Marshall Space Flight Center, Neutral Buoyancy Simulator Facility, Rideout Road, Huntsville, Madison County, AL

  9. Aeronautics and space report of the president, 1974 activities

    NASA Technical Reports Server (NTRS)

    1975-01-01

    The U.S. Government activities for 1974 in aeronautics and space are presented. Significant contributions toward the fulfillment of the nation's goals in space and aeronautics are covered, including application of space systems and technology to beneficial uses on earth, exploration of space and increase of scientific knowledge, development of improved space systems and technology, international cooperation, and advancement of civil and military aeronautics. Also in 1974, space activities in the private sector expanded to provide additional services to the public. The accomplishments are summarized.

  10. International Space Station (ISS) Risk Reduction Activities

    NASA Technical Reports Server (NTRS)

    Fodroci, Michael

    2011-01-01

    As the assembly of the ISS nears completion, it is worthwhile to step back and review some of the actions pursued by the Program in recent years to reduce risk and enhance the safety and health of ISS crewmembers, visitors, and space flight participants. While the ISS requirements and initial design were intended to provide the best practicable levels of safety, it is always possible to reduce risk -- given the determination and commitment to do so. The following is a summary of some of the steps taken by the ISS Program Manager, by our International Partners, by hardware and software designers, by operational specialists, and by safety personnel to continuously enhance the safety of the ISS. While decades of work went into developing the ISS requirements, there are many things in a Program like the ISS that can only be learned through actual operational experience. These risk reduction activities can be divided into roughly three categories: (1) Areas that were initially noncompliant which have subsequently been brought into compliance or near compliance (i.e., Micrometeoroid and Orbital Debris [MMOD] protection, acoustics) (2) Areas where initial design requirements were eventually considered inadequate and were subsequently augmented (i.e., Toxicity Level 4 materials, emergency hardware and procedures) (3) Areas where risks were initially underestimated, and have subsequently been addressed through additional mitigation (i.e., Extravehicular Activity [EVA] sharp edges, plasma shock hazards) Due to the hard work and cooperation of many parties working together across the span of nearly a decade, the ISS is now a safer and healthier environment for our crew, in many cases exceeding the risk reduction targets inherent in the intent of the original design. It will provide a safe and stable platform for utilization and discovery.

  11. Aeronautics and space report of the President, 1982 activities

    NASA Technical Reports Server (NTRS)

    1983-01-01

    Achievements of the space program are summerized in the area of communication, Earth resources, environment, space sciences, transportation, aeronautics, and space energy. Space program activities of the various deprtments and agencies of the Federal Government are discussed in relation to the agencies' goals and policies. Records of U.S. and world spacecraft launchings, successful U.S. launches for 1982, U.S. launched applications and scientific satellites and space probes since 1975, U.S. and Soviet manned spaceflights since 1961, data on U.S. space launch vehicles, and budget summaries are provided. The national space policy and the aeronautical research and technology policy statements are included.

  12. Harnessing CRISPR-Cas9 immunity for genetic engineering.

    PubMed

    Charpentier, Emmanuelle; Marraffini, Luciano A

    2014-06-01

    CRISPR-Cas encodes an adaptive immune system that defends prokaryotes against infectious viruses and plasmids. Immunity is mediated by Cas nucleases, which use small RNA guides (the crRNAs) to specify a cleavage site within the genome of invading nucleic acids. In type II CRISPR-Cas systems, the DNA-cleaving activity is performed by a single enzyme Cas9 guided by an RNA duplex. Using synthetic single RNA guides, Cas9 can be reprogrammed to create specific double-stranded DNA breaks in the genomes of a variety of organisms, ranging from human cells to bacteria, and thus constitutes a powerful tool for genetic engineering. Here we describe recent advancements in our understanding of type II CRISPR-Cas immunity and how these studies led to revolutionary genome editing applications.

  13. Peaceful Use of Outer Space: principles of Japanese Policies on Utilization and Activities in Outer space

    NASA Astrophysics Data System (ADS)

    Kosuge, Toshio

    2002-01-01

    " P e aceful use of outer space of outer space.....Principles of exploitation of outer space was passed in the Japanese Diet. It clearly mentioned that any activity of launching space object into outer space and developing launching rocket should be exclusively for peaceful purpose. NASDA was also established based upon the same principles of the public law. Japanese interpretation of Space Treaty and other related international agreements has been more strict on peaceful use of outer space, like non-military use rather than non-aggressive, because of influence of Japanese Constitution. Treaty and other agreements is analyzed through rapid development of its space activities, technologies and international cooperation with other space powers. Through more than thirty years experiences in space activities in public and private sectors, Japanese domestic laws and policies have not been changed in relation with basic principles. and laws relating to space activities in order to develop new space law and more international cooperation for space utilization rather than military use in new century.

  14. Highly efficient Cas9-mediated transcriptional programming

    DOE PAGES

    Chavez, Alejandro; Scheiman, Jonathan; Vora, Suhani; ...

    2015-03-02

    The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. Here we describe an improved transcriptional regulator through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. Here, we demonstrate its utility in activating endogenous coding and non-coding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).

  15. Telerobotic activities at Johnson Space Center

    NASA Technical Reports Server (NTRS)

    Price, Charles R.

    1989-01-01

    The Johnson Space Center telerobotic efforts span three major thrusts: (1) sustaining and expanding the capability of the Shuttle manipulator; (2) developing and integrating the multiple telerobotic system of the Space Station; and (3) fostering and applying research in all areas of telerobotics technology within the government, private, and academic sectors.

  16. Activities of the Center for Space Construction

    NASA Technical Reports Server (NTRS)

    1993-01-01

    The Center for Space Construction (CSC) at the University of Colorado at Boulder is one of eight University Space Engineering Research Centers established by NASA in 1988. The mission of the center is to conduct research into space technology and to directly contribute to space engineering education. The center reports to the Department of Aerospace Engineering Sciences and resides in the College of Engineering and Applied Science. The college has a long and successful track record of cultivating multi-disciplinary research and education programs. The Center for Space Construction is prominent evidence of this record. At the inception of CSC, the center was primarily founded on the need for research on in-space construction of large space systems like space stations and interplanetary space vehicles. The scope of CSC's research has now evolved to include the design and construction of all spacecraft, large and small. Within this broadened scope, our research projects seek to impact the underlying technological basis for such spacecraft as remote sensing satellites, communication satellites, and other special purpose spacecraft, as well as the technological basis for large space platforms. The center's research focuses on three areas: spacecraft structures, spacecraft operations and control, and regolith and surface systems. In the area of spacecraft structures, our current emphasis is on concepts and modeling of deployable structures, analysis of inflatable structures, structural damage detection algorithms, and composite materials for lightweight structures. In the area of spacecraft operations and control, we are continuing our previous efforts in process control of in-orbit structural assembly. In addition, we have begun two new efforts in formal approach to spacecraft flight software systems design and adaptive attitude control systems. In the area of regolith and surface systems, we are continuing the work of characterizing the physical properties of lunar

  17. Youth activity spaces and daily exposure to tobacco outlets.

    PubMed

    Lipperman-Kreda, Sharon; Morrison, Christopher; Grube, Joel W; Gaidus, Andrew

    2015-07-01

    We explored whether exposure to tobacco outlets in youths' broader activity spaces differs from that obtained using traditional geographic measures of exposure to tobacco outlet within buffers around homes and schools. Youths completed an initial survey, daily text-prompted surveys, and carried GPS-enabled phones for one week. GPS locations were geocoded and activity spaces were constructed by joining sequential points. We calculated the number of tobacco outlets around these polylines and around homes and schools. Results suggest that activity spaces provide a more accurate measure of tobacco outlet exposures than traditional measures. Assessing tobacco outlet exposure within activity spaces may yield significant information to advance the field.

  18. Space station group activities habitability module study

    NASA Technical Reports Server (NTRS)

    Nixon, David

    1986-01-01

    This study explores and analyzes architectural design approaches for the interior of the Space Station Habitability Module (originally defined as Habitability Module 1 in Space Station Reference Configuration Decription, JSC-19989, August 1984). In the Research Phase, architectural program and habitability design guidelines are specified. In the Schematic Design Phase, a range of alternative concepts is described and illustrated with drawings, scale-model photographs and design analysis evaluations. Recommendations are presented on the internal architectural, configuration of the Space Station Habitability Module for such functions as the wardroom, galley, exercise facility, library and station control work station. The models show full design configurations for on-orbit performance.

  19. Individualized Instruction in Science, Earth Space Project, Learning Activities Package.

    ERIC Educational Resources Information Center

    Kuczma, R. M.

    Learning Activity Packages (LAP) relating to the earth and space are presented for use in sampling a new type of learning for a whole year. Eighteen topics are incorporated into five units: (1) introduction to individualized learning, (2) observation versus interpretation, (3) chemistry in the space age, (4) the space age interdisciplines, and (5)…

  20. Physics of Colloids in Space: Microgravity Experiment Launched, Installed, and Activated on the International Space Station

    NASA Technical Reports Server (NTRS)

    Doherty, Michael P.

    2002-01-01

    The Physics of Colloids in Space (PCS) experiment is a Microgravity Fluids Physics investigation that is presently located in an Expedite the Process of Experiments to Space Station (EXPRESS) Rack on the International Space Station. PCS was launched to the International Space Station on April 19, 2001, activated on May 31, 2001, and will continue to operate about 90 hr per week through May 2002.

  1. Space Adaptation of Active Mirror Segment Concepts

    NASA Technical Reports Server (NTRS)

    Ames, Gregory H.

    1999-01-01

    This report summarizes the results of a three year effort by Blue Line Engineering Co. to advance the state of segmented mirror systems in several separate but related areas. The initial set of tasks were designed to address the issues of system level architecture, digital processing system, cluster level support structures, and advanced mirror fabrication concepts. Later in the project new tasks were added to provide support to the existing segmented mirror testbed at Marshall Space Flight Center (MSFC) in the form of upgrades to the 36 subaperture wavefront sensor. Still later, tasks were added to build and install a new system processor based on the results of the new system architecture. The project was successful in achieving a number of important results. These include the following most notable accomplishments: 1) The creation of a new modular digital processing system that is extremely capable and may be applied to a wide range of segmented mirror systems as well as many classes of Multiple Input Multiple Output (MIMO) control systems such as active structures or industrial automation. 2) A new graphical user interface was created for operation of segmented mirror systems. 3) The development of a high bit rate serial data loop that permits bi-directional flow of data to and from as many as 39 segments daisy-chained to form a single cluster of segments. 4) Upgrade of the 36 subaperture Hartmann type Wave Front Sensor (WFS) of the Phased Array Mirror, Extendible Large Aperture (PAMELA) testbed at MSFC resulting in a 40 to 5OX improvement in SNR which in turn enabled NASA personnel to achieve many significant strides in improved closed-loop system operation in 1998. 5) A new system level processor was built and delivered to MSFC for use with the PAMELA testbed. This new system featured a new graphical user interface to replace the obsolete and non-supported menu system originally delivered with the PAMELA system. The hardware featured Blue Line's new stackable

  2. Foreign DNA capture during CRISPR–Cas adaptive immunity

    PubMed Central

    Nuñez, James K.; Harrington, Lucas B.; Kranzusch, Philip J.; Engelman, Alan N.; Doudna, Jennifer A.

    2015-01-01

    Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30–40 base pair (bp) lengths into clustered regularly interspaced short palindromic repeats (CRISPR) loci as spacer segments1–6. The universally conserved Cas1–Cas2 integrase complex catalyzes spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases7–13. How the Cas1–Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1–Cas2 complex bound to cognate 33 nucleotide (nt) protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3′–OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo2–4. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1–Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci. PMID:26503043

  3. Foreign DNA capture during CRISPR-Cas adaptive immunity.

    PubMed

    Nuñez, James K; Harrington, Lucas B; Kranzusch, Philip J; Engelman, Alan N; Doudna, Jennifer A

    2015-11-26

    Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30-40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments. The universally conserved Cas1-Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases. How the Cas1-Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1-Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3'-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1-Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.

  4. Expanding the CRISPR Toolbox: Targeting RNA with Cas13b.

    PubMed

    Barrangou, Rodolphe; Gersbach, Charles A

    2017-02-16

    In this issue of Molecular Cell, Smargon et al. (2017) unearth Cas13b from type VI-B CRISPR-Cas immune systems and characterize its RNA-guided, RNA-targeting activity, including regulation by the novel co-factors Csx27 and Csx28, as well as non-specific collateral RNA damage.

  5. Space Activism as an Epiphanic Belief System

    NASA Technical Reports Server (NTRS)

    Mendell, Wendell

    2006-01-01

    Years of interaction with young people in the space industry and in space activists groups led to my observation that many such individuals can cite a quite specific life event that triggered a life-long interest in or commitment to creating a space future. I am particularly intrigued by parallels between such experiences and the phenomenon of epiphanic experiences among committed Christians. I see analogies between the puzzlement among space activists and among Christian groups as to the reasons for so many people being "unbelievers." At a small international meeting on lunar exploration in 2003, I heard two separate lunch speakers cite such personal experiences. At the beginning of a break in that meeting, I grabbed the microphone from the chairman and asked each person to write down on a pad by his chair whether or not he (or she) had experienced a specific event that led to their involvement in space. If the answer was positive, I asked for a brief narrative, for their age at the time, and for their current age. I received 53 submissions, 20% of which simply stated that their involvement in space exploration was happenstance. (Apollo astronaut John Young was among these.) The other 80% of the submissions had specific stories. The ages at the time of the epiphany ranged from 4 to 47; and their current ages ranged from 22 to 78. I will present a high-level characterization of these inputs. Interest in space exploration as a form of belief system is consistent with choosing NASA goals for the purpose of inspiration and with phenomena such as the "Overview Effect". More research might explore what form the transcendent experience takes and whether it might be associated with feelings of universal connection such as the noosphere or "The Force". From a pragmatic point of view, outreach strategies for exploration should focus on giving individuals access to personal, potentially transformational experiences as opposed to astronaut talks at civic clubs.

  6. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.

    PubMed

    Polstein, Lauren R; Perez-Pinera, Pablo; Kocak, D Dewran; Vockley, Christopher M; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E; Reddy, Timothy E; Gersbach, Charles A

    2015-08-01

    Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function.

  7. Aeronautics and Space Report of the President: 1975 Activities.

    ERIC Educational Resources Information Center

    National Aeronautics and Space Administration, Washington, DC.

    This report, submitted to the Congress by President Ford in accordance with the National Aeronautics and Space Act of 1958, summarizes the United States' space and aeronautics activities for the year 1975. Detailed summaries of the activities of the following governmental departments or agencies are provided: National Aeronautics and Space…

  8. New CRISPR-Cas systems from uncultivated microbes.

    PubMed

    Burstein, David; Harrington, Lucas B; Strutt, Steven C; Probst, Alexander J; Anantharaman, Karthik; Thomas, Brian C; Doudna, Jennifer A; Banfield, Jillian F

    2017-02-09

    CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNA extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.

  9. New CRISPR–Cas systems from uncultivated microbes

    NASA Astrophysics Data System (ADS)

    Burstein, David; Harrington, Lucas B.; Strutt, Steven C.; Probst, Alexander J.; Anantharaman, Karthik; Thomas, Brian C.; Doudna, Jennifer A.; Banfield, Jillian F.

    2016-12-01

    CRISPR–Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR–Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR–Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNA extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR–Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR–Cas system. In bacteria, we discovered two previously unknown systems, CRISPR–CasX and CRISPR–CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.

  10. Active rejection of persistent disturbances in flexible space structures

    NASA Technical Reports Server (NTRS)

    Hwang, Cheng-Neng; Jayasuriya, Suhada; Parlos, Alexander G.; Sunkel, John W.

    1990-01-01

    A dynamic compensator for active rejection of persistent disturbances in flexible space structures is designed on the principle of the H(infinity)-optimization of the sensitivity transfer function matrix. A general state space solution is formulated to the multiinput multioutput H(infinity)-optimal control problem, allowing the use of the H(infinity)-optimal synthesis algorithm for the state-space models of space structures that result from model order reduction. Disturbances encountered in flexible space structures, such as shuttle docking, are investigated using the high-mode and the reduced-order models of a cantilevered two-bay truss, demonstrating the applicability of the H(infinity)-optimal approach.

  11. Qualitative GIS and the Visualization of Narrative Activity Space Data

    PubMed Central

    Mennis, Jeremy; Mason, Michael J.; Cao, Yinghui

    2012-01-01

    Qualitative activity space data, i.e. qualitative data associated with the routine locations and activities of individuals, are recognized as increasingly useful by researchers in the social and health sciences for investigating the influence of environment on human behavior. However, there has been little research on techniques for exploring qualitative activity space data. This research illustrates the theoretical principles of combining qualitative and quantitative data and methodologies within the context of GIS, using visualization as the means of inquiry. Through the use of a prototype implementation of a visualization system for qualitative activity space data, and its application in a case study of urban youth, we show how these theoretical methodological principles are realized in applied research. The visualization system uses a variety of visual variables to simultaneously depict multiple qualitative and quantitative attributes of individuals’ activity spaces. The visualization is applied to explore the activity spaces of a sample of urban youth participating in a study on the geographic and social contexts of adolescent substance use. Examples demonstrate how the visualization may be used to explore individual activity spaces to generate hypotheses, investigate statistical outliers, and explore activity space patterns among subject subgroups. PMID:26190932

  12. Viking mission support. [Deep Space Network activities

    NASA Technical Reports Server (NTRS)

    Johnston, D. W. H.

    1977-01-01

    Statistics listing the Deep Space Network tracking and command support and the discrepancy report status for 1 January through 28 February 1977 are presented in tables. The initial Viking extended mission period of normal DSN support, following the nonstandard operations during the solar conjunction period is included. Operational testing subsequent to the MK III data system installations at DSS 12, 44, and 62 during this period are also discussed.

  13. Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization.

    PubMed

    Senturk, Serif; Shirole, Nitin H; Nowak, Dawid G; Corbo, Vincenzo; Pal, Debjani; Vaughan, Alexander; Tuveson, David A; Trotman, Lloyd C; Kinney, Justin B; Sordella, Raffaella

    2017-02-22

    The CRISPR/Cas9 system is a powerful tool for studying gene function. Here, we describe a method that allows temporal control of CRISPR/Cas9 activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9) enables conditional Cas9 expression and temporal control of gene editing in the presence of an FKBP12 synthetic ligand. This system can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest without co-modulation of the latter. In particular, when co-expressed with inducible Cre-ER(T2), our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with single-cell specificity. We anticipate this platform will be used for the systematic characterization and identification of essential genes, as well as the investigation of the interactions between functional genes.

  14. Calibrated Ancillary System (CAS) user's guide, volume 7

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of CAS. Volume 7 describes the data flow engineer (DFE) user mission planning procedures which include instructions for processing the SDT/TDT (shuttle data tape/telemetry descriptor tape).

  15. Calibrated Ancillary System (CAS) user's guide, volume 5

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of CAS. Volume 5 describes the testing user mission planning procedures including the bulletin board system and ancillary products procedures. Instructions for viewing the SDT/TDT (shuttle data tape/telemetry descriptor tape) data base and the file management menu are also given.

  16. Calibrated Ancillary System (CAS) user's guide, volume 6

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of CAS. Volume 6 describes ancillary products procedures, enhancement menu and processing task procedures for SDT/TDT (shuttle data tape/telemetry descriptor tape), database errors and network data driver (NDD) product menu procedures, and utility menu procedures.

  17. Calibrated Ancillary System (CAS) user's guide, volume 4

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of the CAS. Volume 4 presents the GSFC user mission planning procedures covering the mission planning main menu, bulletin board system, ancillary products menu, utility menu procedures, and ancillary support files procedures.

  18. The Global Space Geodesy Network: Activities Underway

    NASA Astrophysics Data System (ADS)

    Pearlman, Michael R.; Ipatov, Alexander; Long, James; Ma, Chopo; Merkowitz, Stephen; Neilan, Ruth; Noll, Carey; Pavlis, Erricos; Shargorodsky, Victor; Stowers, David; Wetzel, Scott

    2014-05-01

    Several initiatives are underway that should make substantial improvement over the next decade to the international space geodesy network as the international community works toward the GGOS 2020 goal of 32 globally distributed Core Sites with co-located VLBI, SLR, GNSS and DORIS. The Russian Space Agency and the Russian Academy of Sciences are moving forward with an implementation of six additional SLR systems and a number of GNSS receivers to sites outside Russia to expand GNSS tracking and support GGOS. The NASA Space Geodesy program has completed its prototype development phase and is now embarking on an implementation phase that is planning for deployment of 6 - 10 core sites in key geographic locations to support the global network. Additional sites are in the process of implementation in Europe and Asia. Site evaluation studies are in progress, looking at some new potential sites and there are ongoing discussions for partnership arrangements with interested agencies for new sites in South America and Africa. Work continues on the site layout design to avoid RF interference issues among co-located instruments and with external communications and media system. The placement of new and upgraded sites is guided by appropriate Observing System Simulation Experiments (OSSEs) conducted under the support of the interested international agencies. The results will help optimize the global distribution of core geodetic observatories and they will lead to the improvement of the data products from the future network. During this effort it is also recognized that co-located sites with less than the full core complement will continue to play an important and critical role in filling out the global network and strengthening the connection among the techniques. This talk will give an update on the current state of expansion of the global network and the projection for the network configuration that we forecast over the next 10 years.

  19. Aeronautics and space report of the President, 1983 activities

    NASA Technical Reports Server (NTRS)

    1984-01-01

    Achievements in communication; space science; space transportation; aeronautics; and Earth resources and environment are summarized. Activities of the various Federal agencies and cooperation with NASA in these areas are described. The Presidential policy announcement on the endorsement of commercial operation of expendable launch vehicles is included. Tables show, the space activities budget; a historical budget summary, U.S. space launch vehicles; U.S. and Soviet manned spaceflights, 1961 to 1983; U.S. launched space probes, 1975 to 1983; U.S. launched scientific and applications satellites, 1978 to 1983; the U.S. spacecraft record; the world record of space launches successful in attaining Earth orbit or beyond; and successful U.S. launchings for 1983.

  20. Reorganization of the FSU space program and its influence on worldwide space activities

    NASA Astrophysics Data System (ADS)

    Tarasenko, Maksim V.

    1993-10-01

    The paper examines changes in the organization of the former Soviet space program, current program status, and priorities, and analyzes the impact of these changes on world space activities. It is shown that after the breakup of the USSR Russia took over general responsibility for Soviet space activity. Space program management is being reorganized to split military and civil activities and to introduce a system of checks and balances. Attempts are being made to diversify the use of military space systems for civil application, including global environmental problems. Economic problems in the former Soviet republics and political tensions between them force them to search for the cooperation with the West. With a balanced Western response this trend could provide long-term mutual benefits.

  1. Vehicle Engineering Development Activities at the Marshall Space Flight Center

    NASA Technical Reports Server (NTRS)

    Fisher, Mark F.; Champion, Robert H., Jr.

    1999-01-01

    New initiatives in the Space Transportation Directorate at the Marshall Space Flight Center include an emphasis on Vehicle Engineering to enhance the strong commitment to the Directorate's projects in the development of flight hardware and flight demonstrators for the advancement of space transportation technology. This emphasis can be seen in the activities of a newly formed organization in the Transportation Directorate, The Vehicle Subsystems Engineering Group. The functions and type of activities that this group works on are described. The current projects of this group are outlined including a brief description of the status and type of work that the group is performing. A summary section is included to describe future activities.

  2. A simulation system for Space Station extravehicular activity

    NASA Technical Reports Server (NTRS)

    Marmolejo, Jose A.; Shepherd, Chip

    1993-01-01

    America's next major step into space will be the construction of a permanently manned Space Station which is currently under development and scheduled for full operation in the mid-1990's. Most of the construction of the Space Station will be performed over several flights by suited crew members during an extravehicular activity (EVA) from the Space Shuttle. Once fully operational, EVA's will be performed from the Space Station on a routine basis to provide, among other services, maintenance and repair operations of satellites currently in Earth orbit. Both voice recognition and helmet-mounted display technologies can improve the productivity of workers in space by potentially reducing the time, risk, and cost involved in performing EVA. NASA has recognized this potential and is currently developing a voice-controlled information system for Space Station EVA. Two bench-model helmet-mounted displays and an EVA simulation program have been developed to demonstrate the functionality and practicality of the system.

  3. Updates on CCMC Activities and GSFC Space Weather Services

    NASA Technical Reports Server (NTRS)

    Zhengm Y.; Hesse, M.; Kuznetsova, M.; Pulkkinen, A.; Rastaetter, L.; Maddox, M.; Taktakishvili, A.; Berrios, D.; Chulaki, A.; Lee, H.; Macneice, P.; Mays, L.; Mendoza, A. M.; Mullinix, R.

    2011-01-01

    In this presentation, we provide updates on CCMC modeling activities, CCMC metrics and validation studies, and other CCMC efforts. In addition, an overview of GSFC Space Weather Services (a sibling organization to the Community Coordinated Modeling Center) and its products/capabilities will be given. We show how some of the research grade models, if running in an operational mode, can help address NASA's space weather needs by providing forecasting/now casting capabilities of significant space weather events throughout the solar system.

  4. Automated entry technologies for confined space work activities: A survey.

    PubMed

    Botti, Lucia; Ferrari, Emilio; Mora, Cristina

    2017-04-01

    Work in confined spaces poses a significant risk to workers and rescuers involved in the emergency response when an accident occurs. Despite several standards and regulations define the safety requirements for such activities, injuries, and fatalities still occur. Furthermore, the on-site inspections after accidents often reveal that both employers and employees fail to implement safe entry procedures. Removing the risk is possible by avoiding the worker entry, but many activities require the presence of the operator inside the confined space to perform manual tasks. The following study investigates the available technologies for hazardous confined space work activities, e.g., cleaning, inspecting, and maintenance tasks. The aim is to provide a systematic review of the automated solutions for high-risk activities in confined spaces, considering the non-man entry as the most effective confined space safety strategy. Second, this survey aims to provide suggestions for future research addressing the design of new technologies. The survey consists of about 60 papers concerning innovative technologies for confined space work activities. The document review shows that several solutions have been developed and automation can replace the workers for a limited number of hazardous tasks. Several activities still require the manual intervention due to the complex characteristics of confined spaces, e.g., to remove the remains of the automatic cleaning process from the bottom of a tank. The results show that available technologies require more flexibility to adapt to such occupational environments and further research is needed.

  5. CRISPR-Cas Technologies and Applications in Food Bacteria.

    PubMed

    Stout, Emily; Klaenhammer, Todd; Barrangou, Rodolphe

    2017-02-28

    Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form adaptive immune systems that occur in many bacteria and most archaea. In addition to protecting bacteria from phages and other invasive mobile genetic elements, CRISPR-Cas molecular machines can be repurposed as tool kits for applications relevant to the food industry. A primary concern of the food industry has long been the proper management of food-related bacteria, with a focus on both enhancing the outcomes of beneficial microorganisms such as starter cultures and probiotics and limiting the presence of detrimental organisms such as pathogens and spoilage microorganisms. This review introduces CRISPR-Cas as a novel set of technologies to manage food bacteria and offers insights into CRISPR-Cas biology. It primarily focuses on the applications of CRISPR-Cas systems and tools in starter cultures and probiotics, encompassing strain-typing, phage resistance, plasmid vaccination, genome editing, and antimicrobial activity.

  6. Guide RNA engineering for versatile Cas9 functionality

    PubMed Central

    Nowak, Chance M.; Lawson, Seth; Zerez, Megan; Bleris, Leonidas

    2016-01-01

    The Clustered Regularly Interspaced Short Palindromic Repeats system allows a single guide RNA (sgRNA) to direct a protein with combined helicase and nuclease activity to the DNA. Streptococcus pyogenes Cas9 (SpCas9), a CRISPR-associated protein, has revolutionized our ability to probe and edit the human genome in vitro and in vivo. Arguably, the true modularity of the Cas9 platform is conferred through the ease of sgRNA programmability as well as the degree of modifications the sgRNA can tolerate without compromising its association with SpCas9 and function. In this review, we focus on the properties and recent engineering advances of the sgRNA component in Cas9-mediated genome targeting. PMID:27733506

  7. Marshall Space Flight Center ECLSS technology activities

    NASA Technical Reports Server (NTRS)

    Wieland, Paul

    1990-01-01

    Viewgraphs on Environmental Control and Life Support System (ECLSS) technology activities are presented. Topics covered include: analytical development; ECLSS modeling approach; example of water reclamation modeling needs; and hardware development and testing.

  8. NASA SpaceWire Activities/Comments/Recommendations

    NASA Technical Reports Server (NTRS)

    Rakow, Glenn

    2006-01-01

    This viewgraph presentation reviews NASA's activities, and proposes recommendations for the further use of the SpaceWire (SpW). The areas covered in this presentation are: (1) Protocol ID assignment, (2) Protocol development, (3) Plug & Play (PnP), (4) Recommended additions t o SpW protocol and (5) SpaceFibre trade.

  9. CFD Modeling Activities at the NASA Stennis Space Center

    NASA Technical Reports Server (NTRS)

    Allgood, Daniel

    2007-01-01

    A viewgraph presentation on NASA Stennis Space Center's Computational Fluid Dynamics (CFD) Modeling activities is shown. The topics include: 1) Overview of NASA Stennis Space Center; 2) Role of Computational Modeling at NASA-SSC; 3) Computational Modeling Tools and Resources; and 4) CFD Modeling Applications.

  10. Space-Based Astronomy: A Teacher's Guide with Activities.

    ERIC Educational Resources Information Center

    National Aeronautics and Space Administration, Washington, DC.

    This curriculum guide uses hands-on activities to help grade 5-8 students and teachers understand the significance of space-based astronomy--astronomical observations made from outside the Earth's atmosphere. The guide begins with a survey of astronomy-related spacecraft that the National Aeronautics and Space Administration (NASA) has sent into…

  11. Optimization of genome editing through CRISPR-Cas9 engineering.

    PubMed

    Zhang, Jian-Hua; Adikaram, Poorni; Pandey, Mritunjay; Genis, Allison; Simonds, William F

    2016-04-01

    CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats)-Cas9 (CRISPR associated protein 9) has rapidly become the most promising genome editing tool with great potential to revolutionize medicine. Through guidance of a 20 nucleotide RNA (gRNA), CRISPR-Cas9 finds and cuts target protospacer DNA precisely 3 base pairs upstream of a PAM (Protospacer Adjacent Motif). The broken DNA ends are repaired by either NHEJ (Non-Homologous End Joining) resulting in small indels, or by HDR (Homology Directed Repair) for precise gene or nucleotide replacement. Theoretically, CRISPR-Cas9 could be used to modify any genomic sequences, thereby providing a simple, easy, and cost effective means of genome wide gene editing. However, the off-target activity of CRISPR-Cas9 that cuts DNA sites with imperfect matches with gRNA have been of significant concern because clinical applications require 100% accuracy. Additionally, CRISPR-Cas9 has unpredictable efficiency among different DNA target sites and the PAM requirements greatly restrict its genome editing frequency. A large number of efforts have been made to address these impeding issues, but much more is needed to fully realize the medical potential of CRISPR-Cas9. In this article, we summarize the existing problems and current advances of the CRISPR-Cas9 technology and provide perspectives for the ultimate perfection of Cas9-mediated genome editing.

  12. ISODEX: An entry point for developing countries into space activities

    NASA Astrophysics Data System (ADS)

    Skinner, Mark Andrew

    2015-08-01

    Several threads current in the community of international space actors have led to calls at UN COPUOS Scientific & Technical Sub-Committee meetings for enhancing the scientific information available on man-made space objects, whilst fostering international space object data sharing. Growing awareness of the problems of space debris proliferation and space traffic management, especially amongst developing countries and non-traditional space faring nations, have fueled their desires to become involved in the areas of space object tracking, utilizing relatively modest astronomical instrumentation. Additionally, several commercial satellite operators, members of the Satellite Data Association, have called for augmentation of the information available from existing catalogs. This confluence of factors has led to an international discussion, at the UN and elsewhere, of the possibility of creating a clearing-house for parties willing to share data on space objects, with a working title of the “International Space Object Data Exchange” (ISODEX). We discuss the ideas behind this concept, how it might be implemented, and it might enhance the public’s knowledge of space activities, as well as providing an entry point into space for developing countries.

  13. Trajectory Data Analyses for Pedestrian Space-time Activity Study

    PubMed Central

    Qi, Feng; Du, Fei

    2013-01-01

    It is well recognized that human movement in the spatial and temporal dimensions has direct influence on disease transmission1-3. An infectious disease typically spreads via contact between infected and susceptible individuals in their overlapped activity spaces. Therefore, daily mobility-activity information can be used as an indicator to measure exposures to risk factors of infection. However, a major difficulty and thus the reason for paucity of studies of infectious disease transmission at the micro scale arise from the lack of detailed individual mobility data. Previously in transportation and tourism research detailed space-time activity data often relied on the time-space diary technique, which requires subjects to actively record their activities in time and space. This is highly demanding for the participants and collaboration from the participants greatly affects the quality of data4. Modern technologies such as GPS and mobile communications have made possible the automatic collection of trajectory data. The data collected, however, is not ideal for modeling human space-time activities, limited by the accuracies of existing devices. There is also no readily available tool for efficient processing of the data for human behavior study. We present here a suite of methods and an integrated ArcGIS desktop-based visual interface for the pre-processing and spatiotemporal analyses of trajectory data. We provide examples of how such processing may be used to model human space-time activities, especially with error-rich pedestrian trajectory data, that could be useful in public health studies such as infectious disease transmission modeling. The procedure presented includes pre-processing, trajectory segmentation, activity space characterization, density estimation and visualization, and a few other exploratory analysis methods. Pre-processing is the cleaning of noisy raw trajectory data. We introduce an interactive visual pre-processing interface as well as an

  14. Trajectory data analyses for pedestrian space-time activity study.

    PubMed

    Qi, Feng; Du, Fei

    2013-02-25

    It is well recognized that human movement in the spatial and temporal dimensions has direct influence on disease transmission(1-3). An infectious disease typically spreads via contact between infected and susceptible individuals in their overlapped activity spaces. Therefore, daily mobility-activity information can be used as an indicator to measure exposures to risk factors of infection. However, a major difficulty and thus the reason for paucity of studies of infectious disease transmission at the micro scale arise from the lack of detailed individual mobility data. Previously in transportation and tourism research detailed space-time activity data often relied on the time-space diary technique, which requires subjects to actively record their activities in time and space. This is highly demanding for the participants and collaboration from the participants greatly affects the quality of data(4). Modern technologies such as GPS and mobile communications have made possible the automatic collection of trajectory data. The data collected, however, is not ideal for modeling human space-time activities, limited by the accuracies of existing devices. There is also no readily available tool for efficient processing of the data for human behavior study. We present here a suite of methods and an integrated ArcGIS desktop-based visual interface for the pre-processing and spatiotemporal analyses of trajectory data. We provide examples of how such processing may be used to model human space-time activities, especially with error-rich pedestrian trajectory data, that could be useful in public health studies such as infectious disease transmission modeling. The procedure presented includes pre-processing, trajectory segmentation, activity space characterization, density estimation and visualization, and a few other exploratory analysis methods. Pre-processing is the cleaning of noisy raw trajectory data. We introduce an interactive visual pre-processing interface as well as an

  15. Canada s activities on space debris mitigation technologies

    NASA Astrophysics Data System (ADS)

    Nikanpour, D.

    The threat of space debris to space activities is exponentially rising. Canada, as a space-faring nation having significant investment in space and astronauts participating in space missions, has recognized the risks arising from it and has been active as a participant in understanding and mitigate the problem. Since 1992, Canada has been involved with the creation of a sub-committee on space debris under the government's Interdepartmental Committee on Space (ICS) to deal with the policy and international cooperation on space debris. On the research front, Canadian Space Agency (CSA) has been coordinating the related researches within Canada. This paper outlines the major Canadian research activities on space debris and mitigation technologies along with CSA's future plan on the subject. Canadian research activities on space debris are in 3 major areas: (1) Measurement and modeling of space debris: The work has been led by the CSA (Space Technologies) with participations from research institutes and universities. The experiments cover the analysis and computational modeling of the space debris flux at orbital altitudes of interest for space activities. (2) Space debris mitigation: The technology for mitigating space debris is of key research interest and measures have been taken in the design and launch of LEO earth observation spacecraft, such as RADARSAT. RADARSAT-1, launched in 1995 and still operating, was one of the first commercial spacecraft to consider the effect of orbital debris in its design. Not only was the spacecraft designed to withstand a possible impact on orbit, and not be a source of debris from latches and tie-down mechanisms, but the launch of RADARSAT-1 was also delayed by 25 seconds in a very tight launch window, to avoid a possible impact on orbit. The design for the follow-on RADARSAT-2 spacecraft includes features to protect its Synthetic Aperture Radar (SAR) antenna against possible impact damage due to space debris as well as include

  16. Defining filled and empty space: reassessing the filled space illusion for active touch and vision.

    PubMed

    Collier, Elizabeth S; Lawson, Rebecca

    2016-09-01

    In the filled space illusion, an extent filled with gratings is estimated as longer than an equivalent extent that is apparently empty. However, researchers do not seem to have carefully considered the terms filled and empty when describing this illusion. Specifically, for active touch, smooth, solid surfaces have typically been used to represent empty space. Thus, it is not known whether comparing gratings to truly empty space (air) during active exploration by touch elicits the same illusionary effect. In Experiments 1 and 2, gratings were estimated as longer if they were compared to smooth, solid surfaces rather than being compared to truly empty space. Consistent with this, Experiment 3 showed that empty space was perceived as longer than solid surfaces when the two were compared directly. Together these results are consistent with the hypothesis that, for touch, the standard filled space illusion only occurs if gratings are compared to smooth, solid surfaces and that it may reverse if gratings are compared to empty space. Finally, Experiment 4 showed that gratings were estimated as longer than both solid and empty extents in vision, so the direction of the filled space illusion in vision was not affected by the nature of the comparator. These results are discussed in relation to the dual nature of active touch.

  17. Brain in Space: A Teacher's Guide with Activities for Neuroscience

    NASA Technical Reports Server (NTRS)

    Sullivan, Walter W., Jr.

    1998-01-01

    The lessons and activities in this guide will engage your students in the excitement of space life science investigations after the Neurolab Spacelab mission. It is the authors' goal that the information in this guide will inspire both you and your students to become interested and active participants in this space mission. Few experiences can compare with the excitement and thrill of watching a Shuttle launch. This guide provides an opportunity for you and your students to go one step further by conducting the experiments on Earth that are relevent to the research conducted in space.

  18. The role of Cas8 in type I CRISPR interference.

    PubMed

    Cass, Simon D B; Haas, Karina A; Stoll, Britta; Alkhnbashi, Omer S; Sharma, Kundan; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita; Bolt, Edward L

    2015-05-05

    CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use 'cascade' [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5-Cas7-crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA.

  19. Study of Eclipsing Binary and Multiple Systems in OB Associations IV: Cas OB6 Member DN Cas

    NASA Astrophysics Data System (ADS)

    Bakış, V.; Bakış, H.; Bilir, S.; Eker, Z.

    2016-09-01

    An early-type, massive, short-period (Porb=2d.310951) eclipsing spectroscopic binary DN Cas has been re-visited with new spectral and photometric data. The masses and radii of the components have been obtained as M1=19.04± 0.07 M⊙, M2=13.73± 0.05 M⊙ and R1=7.22± 0.06 R⊙, R2=5.79± 0.06 R⊙, respectively. Both components present synchronous rotation (Vrot1=160 km s-1, Vrot2=130 km s-1) with their orbit. Orbital period analysis yielded a physically bound additional component in the system with a minimum mass of M3=0.88 M⊙ orbiting in an eccentric orbit (e = 0.37 ± 0.2) with an orbital period of P 12 = 42 ± 9 yr. High precision absolute parameters of the system allowed us to derive a distance to DN Cas as 1.7 ± 0.2 kpc which locates the system within the borders of the Cas OB6 association (d = 1.8 kpc). The space velocities and the age of DN Cas are in agreement with those of Cas OB6. The age of DN Cas (τ = 3-5 Myr) is found to be 1-2 Myr older than the embedded clusters (IC 1795, IC 1805, and IC 1848) in the Cas OB6 association, which implies a sequential star formation in the association.

  20. Multilayer active shell mirrors for space telescopes

    NASA Astrophysics Data System (ADS)

    Steeves, John; Jackson, Kathryn; Pellegrino, Sergio; Redding, David; Wallace, J. Kent; Bradford, Samuel Case; Barbee, Troy

    2016-07-01

    A novel active mirror technology based on carbon fiber reinforced polymer (CFRP) substrates and replication techniques has been developed. Multiple additional layers are implemented into the design serving various functions. Nanolaminate metal films are used to provide a high quality reflective front surface. A backing layer of thin active material is implemented to provide the surface-parallel actuation scheme. Printed electronics are used to create a custom electrode pattern and flexible routing layer. Mirrors of this design are thin (< 1.0 mm), lightweight (2.7 kg/m2), and have large actuation capabilities. These capabilities, along with the associated manufacturing processes, represent a significant change in design compared to traditional optics. Such mirrors could be used as lightweight primaries for small CubeSat-based telescopes or as meter-class segments for future large aperture observatories. Multiple mirrors can be produced under identical conditions enabling a substantial reduction in manufacturing cost and complexity. An overview of the mirror design and manufacturing processes is presented. Predictions on the actuation performance have been made through finite element simulations demonstrating correctabilities on the order of 250-300× for astigmatic modes with only 41 independent actuators. A description of the custom metrology system used to characterize the active mirrors is also presented. The system is based on a Reverse Hartmann test and can accommodate extremely large deviations in mirror figure (> 100 μm PV) down to sub-micron precision. The system has been validated against several traditional techniques including photogrammetry and interferometry. The mirror performance has been characterized using this system, as well as closed-loop figure correction experiments on 150 mm dia. prototypes. The mirrors have demonstrated post-correction figure accuracies of 200 nm RMS (two dead actuators limiting performance).

  1. Space Sciences in the classroom: Educational activities of the European Space Agency

    NASA Astrophysics Data System (ADS)

    Korakitis, R.

    2012-01-01

    Education is among the basic, mandatory activities of the European Space Agency (ESA) and aims at all educational levels, from primary school to post-graduate. The primary objective of the ESA educational activities is to enhance the literacy of young people in science and technology and to stimulate interest in STEM (Science - Technology - Engineering - Mathematics) studies and careers, using Space as a theme. The activities mostly follow the IBSE paradigm (Inquiry-Based Science Education) and also aim at the Continuous Professional Development (CPD) for teachers. The backbone supporting all educational activities is the enormous expertise of ESA in the various aspects of Space Science and Technology, like Earth Observation, Space Science (including Astronomy & Astrophysics), Human Spaceflight and Space Technology (launchers, navigation, telecommunications etc.). All educational activities, which are coordinated by the ESA Education Office, are designed for specific age groups, strive to keep the educational community informed and to provide inspirational materials for teachers and students. They can be subdivided in categories, like: Hands-on-projects, opportunities for students, support to teachers, international cooperation activities and outreach initiatives. In addition, ESA develops a variety of educational materials to support teachers in the classroom, both in classic form or on-line, through a network of dedicated websites.

  2. Perspectives from space: NASA classroom information and activities

    NASA Technical Reports Server (NTRS)

    1992-01-01

    This booklet contains the information and classroom activities included on the backs of the eight poster series, 'Perspectives From Space'. The first series, Earth, An Integrated System, contains information on global ecology, remote sensing from space, data products, earth modeling, and international environmental treaties. The second series, Patterns Among Planets, contains information on the solar system, planetary processes, impacts and atmospheres, and a classroom activity on Jupiter's satellite system. The third series, Our Place In The Cosmos, contains information on the scale of the universe, origins of the universe, mission to the universe, and three classroom activities. The fourth series, Our Sun, The Nearest Star, contains information on the Sun. The fifth series, Oasis Of Life, contains information on the development of life, chemical and biological evolution on Earth and the search for other life in the universe. The sixth series, The Influence Of Gravity, contains information on Newton's Law of Gravity, space and microgravity, microgravity environment, and classroom activities on gravity. The seventh series, The Spirit Of Exploration, contains information on space exploration, the Apollo Program, future exploration activities, and two classroom activities. The eighth series, Global Cooperation, contains information on rocketry, the space race, and multi-nation exploration projects.

  3. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

    PubMed Central

    Isaac, R Stefan; Jiang, Fuguo; Doudna, Jennifer A; Lim, Wendell A; Narlikar, Geeta J; Almeida, Ricardo

    2016-01-01

    The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo. DOI: http://dx.doi.org/10.7554/eLife.13450.001 PMID:27130520

  4. Large Active Retrodirective Arrays for Space Applications

    NASA Technical Reports Server (NTRS)

    Chernoff, R. C.

    1978-01-01

    An active retrodirective array (ARA) electronically points a microwave beam back at the apparent source of an incident pilot signal. Retrodirectivity is the result of phase conjugation of the pilot signal received by each element of the array. The problem of supplying the correct phase reference to the phase conjugation circuit (PCC) associated with each element of the array is solved by central phasing. By eliminating the need for structural rigidity, central phasing confers a decisive advantage on ARA's as large spaceborne antennas. A new form of central phasing suitable for very large arrays is described. ARA's may easily be modified to serve both as transmitting and receiving arrays simultaneously. Two new kinds of exact, frequency translating PCC's are described. Such PCC's provide the ARA with input-output isolation and freedom from squint. The pointing errors caused by the radial and transverse components of the ARA's velocity, by the propagation medium, and by multipath are discussed. A two element ARA breadboard was built and tested at JPL. Its performance is limited primarily by multipath induced errors.

  5. Russian space agency activities on the problem of technogenic space debris

    NASA Astrophysics Data System (ADS)

    Blagun, V. P.; Kulik, S. V.; Lukyashchenko, V. I.

    1999-01-01

    In this paper the main directions and the major results of the activities on the problem of technogenic near-earth space (NES) orbital debris are discussed. With regard to monitoring the NES debris environment, the following issues are considered: the catalogue of space objects which includes objects in the geostationary ring, orbital debris models, and ground- and space-based observations. For the protection of spacecraft and Space Station from debris particles multilayer and other shields are used, as well as avoidance manoeuvres. Important issues are the determination of the location of impacts and restoration of the station wall tightness. The BUFFER program has been developed for the risk assessment of impacts of orbital debris particles with the Space Station. Measures are taken to reduce technogenic pollution of NES which include those to prevent launch vehicles and spacecraft explosions. Special attention is placed on the safe utilization of the geostationary orbit. From the results of these studies regulatory documents are issued.

  6. Development of magnetostrictive active members for control of space structures

    NASA Technical Reports Server (NTRS)

    Johnson, Bruce G.; Avakian, Kevin M.; Fenn, Ralph C.; Gaffney, Monique S.; Gerver, Michael J.; Hawkey, Timothy J.; Boudreau, Donald J.

    1992-01-01

    The goal of this Phase 2 Small Business Innovative Research (SBIR) project was to determine the technical feasibility of developing magnetostrictive active members for use as truss elements in space structures. Active members control elastic vibrations of truss-based space structures and integrate the functions of truss structure element, actively controlled actuator, and sensor. The active members must control structural motion to the sub-micron level and, for many proposed space applications, work at cryogenic temperatures. Under this program both room temperature and cryogenic temperature magnetostrictive active members were designed, fabricated, and tested. The results of these performance tests indicated that room temperature magnetostrictive actuators feature higher strain, stiffness, and force capability with lower amplifier requirements than similarly sized piezoelectric or electrostrictive active members, at the cost of higher mass. Two different cryogenic temperature magnetostrictive materials were tested at liquid nitrogen temperatures, both with larger strain capability than the room temperature magnetostrictive materials. The cryogenic active member development included the design and fabrication of a cryostat that allows operation of the cryogenic active member in a space structure testbed.

  7. Overview of materials processing in space activity at Marshall Space Flight Center

    NASA Technical Reports Server (NTRS)

    Williams, J. R.; Chassay, R. P.; Moore, W. W.; Ruff, R. C.; Yates, I. C.

    1984-01-01

    An overview of activities involving the Space Transportation System (STS), now in the operational phase, and results of some of the current space experiments, as well as future research opportunities in microgravity environment, are presented. The experiments of the Materials Processing in Space Program flown on the STS, such as bioseparation processes, isoelectric focusing, solidification and crystal growth processes, containerless processes, and the Materials Experiment Assembly experiments are discussed. Special consideration is given to the experiments to be flown aboard the Spacelab 3 module, the Fluids Experiments System, and the Vapor Crystal Growth System. Ground-based test facilities and planned space research facilities, as well as the nature of the commercialization activities, are briefly explained.

  8. Activities report of the Institute of Space Physics

    NASA Astrophysics Data System (ADS)

    Eliasson, Lars

    Activities at the Swedish Institute of Space Physics (IRF), whose main task is to conduct research and perform observatory measurements in the field of space physics, are addressed. Additionally, the IRF also provides education in space physics. Facts about the IRF and its board, staff, finances, and personnel are given. The research carried out in the four divisions is reviewed. The research covers the following topics: hot magnetospheric plasma investigations; research in the field of mechanical waves, especially concerning infrasound and vibration; space plasma theory, with an emphasis on plasma waves and wave particle interactions in the magnetosphere; experimental studies of waves and wave interactions in space plasmas; and electrodynamic processes related to ionosphere magnetosphere coupling. The observatory program which carried out ground based observations in Kiruna, Lycksele, and Uppsala (Sweden) is reviewed, as is the educational program. Committee appointments, participation in conferences and contributions, and publication and reports of 1991 are also addessed.

  9. Assessing Built Environment Walkability using Activity-Space Summary Measures

    PubMed Central

    Tribby, Calvin P.; Miller, Harvey J.; Brown, Barbara B.; Werner, Carol M.; Smith, Ken R.

    2015-01-01

    There is increasing emphasis on active transportation, such as walking, in transportation planning as a sustainable form of mobility and in public health as a means of achieving recommended physical activity and better health outcomes. A research focus is the influence of the built environment on walking, with the ultimate goal of identifying environmental modifications that invite more walking. However, assessments of the built environment for walkability are typically at a spatially disaggregate level (such as street blocks) or at a spatially aggregate level (such as census block groups). A key issue is determining the spatial units for walkability measures so that they reflect potential walking behavior. This paper develops methods for assessing walkability within individual activity spaces: the geographic region accessible to an individual during a given walking trip. We first estimate street network-based activity spaces using the shortest path between known trip starting/ending points and a travel time budget that reflects potential alternative paths. Based on objective walkability measures of the street blocks, we use three summary measures for walkability within activity spaces: i) the average walkability score across block segments (representing the general level of walkability in the activity space); ii) the standard deviation (representing the walkability variation), and; iii) the network autocorrelation (representing the spatial coherence of the walkability pattern). We assess the method using data from an empirical study of built environment walkability and walking behavior in Salt Lake City, Utah, USA. We visualize and map these activity space summary measures to compare walkability among individuals’ trips within their neighborhoods. We also compare summary measures for activity spaces versus census block groups, with the result that they agree less than half of the time. PMID:27213027

  10. New Space Weather Activities in the World Meteorological Organization

    NASA Astrophysics Data System (ADS)

    Bogdan, Thomas J.; Onsager, Terrance G.

    2010-10-01

    A new era of enhanced international cooperation in space weather operations has begun with the recent initiation of space weather activities within the World Meteorological Organization (WMO), an agency of the United Nations (U.N.) with a membership of 189 states and territories. These activities aim to standardize and enhance space weather observations and data exchange, coordinate end products and services, and foster dialogue between the research and operational communities. The WMO's role is to foster collaboration among the meteorological and hydrological (and now space weather) service providers and to promote the establishment of networks for making and exchanging geophysical observations and the standardization of data and metadata. It also contributes to policy making and has a lead role in efforts to monitor and protect the environment.

  11. Aeronautics and space report of the President, 1980 activities

    NASA Technical Reports Server (NTRS)

    1981-01-01

    The year's achievements in the areas of communication, Earth resources, environment, space sciences, transportation, and space energy are summarized and current and planned activities in these areas at the various departments and agencies of the Federal Government are summarized. Tables show U.S. and world spacecraft records, spacecraft launchings for 1980, and scientific payload anf probes launched 1975-1980. Budget data are included.

  12. Mechanical research and development activities at the European space agency

    NASA Astrophysics Data System (ADS)

    Stavrinidis, C.

    1996-02-01

    The research and development activities of satellite mechanical systems at ESA are driven by the requirements of future European space programmes, evolution of technologies resulting in new technical capabilities, and the need to reduce the cost, to increase the reliability of the European space effort and to improve the competitiveness of the European space industry. Technology developments require in many cases several years from initial concept to technological readiness, and this needs to be taken into account when considering satellite mission requirements. On the other hand difficulties encountered with the performance of existing mechanical system need to be resolved in a shorter timescale. Agency research and development activities of mechanical systems include: - new materials applications - design and manufacture techniques - structural dynamics and low disturbance environment - high precision reflectors - vibroacoustics - meteoroid and debris protection - tribology - pyrotechnics The activities take into account requirements of future space missions for science, earth observation, telecommunications, launchers, and microgravity applications. There are efforts to improve the competitive edge of space industry for example through the European Coordination for Space Standardization (ECSS).

  13. Determination Of The Activity Space By The Stereometric Method

    NASA Astrophysics Data System (ADS)

    Deloison, Y.; Crete, N.; Mollard, R.

    1980-07-01

    To determine the activity space of a sitting subject, it is necessary to go beyond the mere statistical description of morphology and the knowledge of the displacement volume. An anlysis of the positions or variations of the positions of the diverse segmental elements (arms, hands, lower limbs, etc...) in the course of a given activity is required. Of the various methods used to locate quickly and accurately the spatial positions of anatomical points, stereometry makes it possible to plot the three-dimensional coordinates of any point in space in relation to a fixed trirectangle frame of reference determined by the stereome-tric measuring device. Thus, regardless of the orientation and posture of the subject, his segmental elements can be easily pin-pointed, throughout the experiment, within the space they occupy. Using this method, it is possible for a sample of operators seated at an operation station and applying either manual controls or pedals and belonging to a population statistically defined from the data collected and the analyses produced by the anthropometric study to determine a contour line of reach capability marking out the usable working space and to know, within this working space, a contour line of preferential activity that is limited, in space, by the whole range of optimal reach capability of all the subjects.

  14. Activities on space debris in U.S.

    NASA Astrophysics Data System (ADS)

    Johnson, Nicholas L.

    2001-10-01

    In the U.S. space debris activities are addressed at all government levels, from the Executive Office of the President to the individual federal agencies to specialized centers, laboratories, organizations, and research groups. U.S. Space Policy specifically challenges government agencies to seek to minimize the creation of space debris and to promote debris minimization practices, both domestically and internationally. A set of space debris mitigation standard practices has been developed and adopted by relevant U.S. government agencies, and their application by the commercial aerospace community is highly encouraged. A growing number of U.S. government agencies have issued their own space debris mitigation policies, directives, regulations, and standards. Space debris research, including the definition and modeling of the current and future near-Earth space environment and the development of debris protection technologies, is principally conducted by NASA and the Department of Defense. The U.S. Space Surveillance Network continues to provide the most complete and timely characterization of the population of space debris larger than 10 cm. During the past several years major advancements have been achieved in extending this environment definition in LEO to include particles as small as only a few millimeters. The inspection of returned spacecraft surfaces continues to shed light on the even smaller debris population. With improvements in computer technology, new and more capable programs have been and are being developed to solve a number of operational and research problems. Finally, the academic and industrial sectors of the U.S. are also increasing their participation in and contributions to space debris operations and research. The cooperation of spacecraft and launch vehicle developers and operators is essential to the U.S. objective of promoting the preservation of the space environment for future generations.

  15. Activities on Space Debris in U.S.

    NASA Technical Reports Server (NTRS)

    Johnson, Nicholas L.

    2001-01-01

    In the U.S. space debris activities are addressed at all government levels, from the Executive Office of the President to the individual federal agencies to specialized centers, laboratories, organizations, and research groups. U.S. Space Policy specifically challenges government agencies to seek to minimize the creation of space debris and to promote debris minimization practices both domestically and internationally. A set of space debris mitigation standard practices has been developed and adopted by relevant US government agencies, and their application by the commercial aerospace community is highly encouraged. A growing number of US government agencies have issued their own space debris mitigation policies, directives, regulations, and standards. Space debris research, including the definition and modeling of the current and future near-Earth space environment and the development of debris protection technologies, is principally conducted by NASA and the Department of Defense. The U.S. Space Surveillance Network continues to provide the most complete and timely characterization of the population of space debris larger than 10 cm. During the past several years major advancements have been achieved in extending this environment definition in LEO to include particles as small as only a few millimeters. The inspection of returned spacecraft surfaces continues to shed light on the even smaller debris population. With improvements in computer technology, new and more capable programs have been and are being developed to solve a number of operational and research problems. Finally, the academic and industrial sectors of the U.S. are also increasing their participation in and contributions to space debris operations and research. The cooperation of satellite and launch vehicle developers and operators is essential to the U.S. objective of promoting the preservation of the space environment for future generations.

  16. Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering.

    PubMed

    Nuñez, James K; Harrington, Lucas B; Doudna, Jennifer A

    2016-03-18

    The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.

  17. Primary processing of CRISPR RNA by the endonuclease Cas6 in Staphylococcus epidermidis.

    PubMed

    Wakefield, Noelle; Rajan, Rakhi; Sontheimer, Erik J

    2015-10-07

    In many bacteria and archaea, an adaptive immune system (CRISPR-Cas) provides immunity against foreign genetic elements. This system uses CRISPR RNAs (crRNAs) derived from the CRISPR array, along with CRISPR-associated (Cas) proteins, to target foreign nucleic acids. In most CRISPR systems, endonucleolytic processing of crRNA precursors (pre-crRNAs) is essential for the pathway. Here we study the Cas6 endonuclease responsible for crRNA processing in the Type III-A CRISPR-Cas system from Staphylococcus epidermidis RP62a, a model for Type III-A CRISPR-Cas systems, and define substrate requirements for SeCas6 activity. We find that SeCas6 is necessary and sufficient for full-length crRNA biogenesis in vitro, and that it relies on both sequence and stem-loop structure in the 3' half of the CRISPR repeat for recognition and processing.

  18. Aeronautics and Space Report of the President: Fiscal Year 1996 Activities

    NASA Technical Reports Server (NTRS)

    1996-01-01

    Topics considered include: (1) Space launch activities: space shuttle missions; expendable launch vehicles. (2) Space science: astronomy and space physics; solar system exploration. (3) Space flight and technology: life and microgravity sciences; space shuttle technology; reuseable launch vehicles; international space station; energy; safety and mission assurance; commercial development and regulation of space; surveillance. (4) Space communications: communications satellites; space network; ground networks; mission control and data systems. (5) Aeronautical activities: technology developments; air traffic control and navigation; weather-related aeronautical activities; flight safety and security; aviation medicine and human factors. (6) Studies of the planet earth: terrestrial studies and applications: atmospheric studies: oceanographic studies; international aeronautical and space activities; and appendices.

  19. Changes in gastric myoelectric activity during space flight

    NASA Technical Reports Server (NTRS)

    Harm, Deborah L.; Sandoz, Gwenn R.; Stern, Robert M.

    2002-01-01

    The purpose of the present study was to examine postprandial myoelectric activity of the stomach and gastric activity associated with space motion sickness using electrogastrography. Three crewmembers participated in this investigation. Preflight, subjects exhibited normal postprandial responses to the ingestion of a meal. Inflight, crewmembers exhibited an abnormal decrease in the power of the normal gastric slow wave after eating on flight day 1, but had a normal postprandial response by flight day 3. Prior to and during episodes of nausea and vomiting, the electrical activity of the stomach became dysrhythmic with 60-80% of the spectral power in the bradygastric and tachygastric frequency ranges. These findings indicate that gastric motility may be decreased during the first few days of space flight. In addition, changes in the frequency of the gastric slow wave associated with space motion sickness symptoms are consistent with those reported for laboratory-induced motion sickness.

  20. Neuromuscular activation patterns during treadmill walking after space flight

    NASA Technical Reports Server (NTRS)

    Layne, C. S.; McDonald, P. V.; Bloomberg, J. J.

    1997-01-01

    Astronauts adopt a variety of neuromuscular control strategies during space flight that are appropriate for locomoting in that unique environment, but are less than optimal upon return to Earth. We report here the first systematic investigation of potential adaptations in neuromuscular activity patterns associated with postflight locomotion. Astronaut-subjects were tasked with walking on a treadmill at 6.4 km/h while fixating a visual target 30 cm away from their eyes after space flights of 8-15 days. Surface electromyography was collected from selected lower limb muscles and normalized with regard to mean amplitude and temporal relation to heel strike. In general, high correlations (more than 0.80) were found between preflight and postflight activation waveforms for each muscle and each subject: however relative activation amplitude around heel strike and toe off was changed as a result of flight. The level of muscle cocontraction and activation variability, and the relationship between the phasic characteristics of the ankle musculature in preparation for toe off also were altered by space flight. Subjects also reported oscillopsia during treadmill walking after flight. These findings indicate that, after space flight, the sensory-motor system can generate neuromuscular-activation strategies that permit treadmill walking, but subtle changes in lower-limb neuromuscular activation are present that may contribute to increased lower limb kinematic variability and oscillopsia also present during postflight walking.

  1. Characterization and evolution of Salmonella CRISPR-Cas systems.

    PubMed

    Shariat, Nikki; Timme, Ruth E; Pettengill, James B; Barrangou, Rodolphe; Dudley, Edward G

    2015-02-01

    Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide adaptive immunity from invasive genetic elements and encompass three essential features: (i) cas genes, (ii) a CRISPR array composed of spacers and direct repeats and (iii) an AT-rich leader sequence upstream of the array. We performed in-depth sequence analysis of the CRISPR-Cas systems in >600 Salmonella, representing four clinically prevalent serovars. Each CRISPR-Cas feature is extremely conserved in the Salmonella, and the CRISPR1 locus is more highly conserved than CRISPR2. Array composition is serovar-specific, although no convincing evidence of recent spacer acquisition against exogenous nucleic acids exists. Only 12% of spacers match phage and plasmid sequences and self-targeting spacers are associated with direct repeat variants. High nucleotide identity (>99.9%) exists across the cas operon among isolates of a single serovar and in some cases this conservation extends across divergent serovars. These observations reflect historical CRISPR-Cas immune activity, showing that this locus has ceased undergoing adaptive events. Intriguingly, the high level of conservation across divergent serovars shows that the genetic integrity of these inactive loci is maintained over time, contrasting with the canonical view that inactive CRISPR loci degenerate over time. This thorough characterization of Salmonella CRISPR-Cas systems presents new insights into Salmonella CRISPR evolution, particularly with respect to cas gene conservation, leader sequences, organization of direct repeats and protospacer matches. Collectively, our data suggest that Salmonella CRISPR-Cas systems are no longer immunogenic; rather, their impressive conservation indicates they may have an alternative function in Salmonella.

  2. Characterization and evolution of Salmonella CRISPR-Cas systems.

    PubMed

    Shariat, Nikki; Timme, Ruth E; Pettengill, James B; Barrangou, Rodolphe; Dudley, Edward G

    2015-02-01

    Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide adaptive immunity from invasive genetic elements and encompass three essential features: (i) cas genes, (ii) a CRISPR array composed of spacers and direct repeats and (iii) an AT-rich leader sequence upstream of the array. We performed in-depth sequence analysis of the CRISPR-Cas systems in >600 Salmonella, representing four clinically prevalent serovars. Each CRISPR-Cas feature is extremely conserved in the Salmonella, and the CRISPR1 locus is more highly conserved than CRISPR2. Array composition is serovar-specific, although no convincing evidence of recent spacer acquisition against exogenous nucleic acids exists. Only 12 % of spacers match phage and plasmid sequences and self-targeting spacers are associated with direct repeat variants. High nucleotide identity (>99.9 %) exists across the cas operon among isolates of a single serovar and in some cases this conservation extends across divergent serovars. These observations reflect historical CRISPR-Cas immune activity, showing that this locus has ceased undergoing adaptive events. Intriguingly, the high level of conservation across divergent serovars shows that the genetic integrity of these inactive loci is maintained over time, contrasting with the canonical view that inactive CRISPR loci degenerate over time. This thorough characterization of Salmonella CRISPR-Cas systems presents new insights into Salmonella CRISPR evolution, particularly with respect to cas gene conservation, leader sequences, organization of direct repeats and protospacer matches. Collectively, our data suggest that Salmonella CRISPR-Cas systems are no longer immunogenic; rather, their impressive conservation indicates they may have an alternative function in Salmonella.

  3. Destinations That Older Adults Experience Within Their GPS Activity Spaces Relation to Objectively Measured Physical Activity

    PubMed Central

    Winters, Meghan; Ashe, Maureen C.; Clarke, Philippa; McKay, Heather

    2016-01-01

    Identifying the relevant geography is an ongoing obstacle to effectively evaluate the influence of neighborhood built environment on physical activity. We characterized density and diversity of destinations that 77 older adults experienced within individually representative GPS activity spaces and traditional residential buffers and assessed their associations with accelerometry-measured physical activity. Traditional residential buffers had lower destination density and diversity than activity spaces. Activity spaces based only on pedestrian and bicycling trips had higher destination densities than all-mode activity spaces. Regardless of neighborhood definition, adjusted associations between destinations and physical activity generally failed to reach statistical significance. However, within pedestrian and bicycling-based activity spaces each additional destination type was associated with 243.3 more steps/day (95% confidence interval (CI) 36.0, 450.7). Traditional buffers may not accurately portray the geographic space or neighborhood resources experienced by older adults. Pedestrian and bicycling activity spaces elucidate the importance of destinations for facilitating active transportation. PMID:26783370

  4. Nucleosomes impede Cas9 access to DNA in vivo and in vitro

    PubMed Central

    Horlbeck, Max A; Witkowsky, Lea B; Guglielmi, Benjamin; Replogle, Joseph M; Gilbert, Luke A; Villalta, Jacqueline E; Torigoe, Sharon E; Tjian, Robert; Weissman, Jonathan S

    2016-01-01

    The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties. DOI: http://dx.doi.org/10.7554/eLife.12677.001 PMID:26987018

  5. Collaborative Human Engineering Work in Space Exploration Extravehicular Activities (EVA)

    NASA Technical Reports Server (NTRS)

    DeSantis, Lena; Whitmore, Mihriban

    2007-01-01

    A viewgraph presentation on extravehicular activities in space exploration in collaboration with other NASA centers, industries, and universities is shown. The topics include: 1) Concept of Operations for Future EVA activities; 2) Desert Research and Technology Studies (RATS); 3) Advanced EVA Walkback Test; 4) Walkback Subjective Results; 5) Integrated Suit Test 1; 6) Portable Life Support Subsystem (PLSS); 7) Flex PLSS Design Process; and 8) EVA Information System; 9)

  6. Active vibration damping of the Space Shuttle remote manipulator system

    NASA Technical Reports Server (NTRS)

    Scott, Michael A.; Gilbert, Michael G.; Demeo, Martha E.

    1991-01-01

    The feasibility of providing active damping augmentation of the Space Shuttle Remote Manipulator System (RMS) following normal payload handling operations is investigated. The approach used in the analysis is described, and the results for both linear and nonlinear performance analysis of candidate laws are presented, demonstrating that significant improvement in the RMS dynamic response can be achieved through active control using measured RMS tip acceleration data for feedback.

  7. Active vibration damping of the Space Shuttle Remote Manipulator System

    NASA Technical Reports Server (NTRS)

    Scott, Michael A.; Gilbert, Michael G.; Demeo, Martha E.

    1991-01-01

    The feasibility of providing active damping augmentation of the Space Shuttle Remote Manipulator System (RMS) following normal payload-handling operations is investigated. The approach used in the analysis is described and the results from both linear and nonlinear performance analyses of candidate laws are presented, demonstrating that significant improvement in the RMS dynamic response can be achieved through active control using measured RMS tip acceleration data for feedback.

  8. Six degree of freedom active vibration damping for space application

    NASA Technical Reports Server (NTRS)

    Haynes, Leonard S.

    1993-01-01

    Work performed during the period 1 Jan. - 31 Mar. 1993 on six degree of freedom active vibration damping for space application is presented. A performance and cost report is included. Topics covered include: actuator testing; mechanical amplifier design; and neural network control system development and experimental evaluation.

  9. Draft position paper on knowledge management in space activities

    NASA Technical Reports Server (NTRS)

    Holm, Jeanne; Moura, Denis

    2003-01-01

    As other fields of industry, space activities are facing the challenge of Knowledge Management and the International Academy of Astronautics decided to settle in 2002 a Study Group to analyse the problem and issue general guidelines. This communication presents the draft position paper of this group in view to be discussed during the 2003 IAF Congress.

  10. Space Station Freedom. An Activity Book for Elementary School Students.

    ERIC Educational Resources Information Center

    National Aeronautics and Space Administration, Washington, DC.

    This booklet was prepared by the National Aeronautics and Space Administration (NASA) for use by teachers in the classroom or by parents at home. The descriptions, classroom activities and illustrations are designed for elementary-level school children. On each right-hand page is a simple line drawing that illustrates the narrative and the…

  11. Activity Spaces and Urban Adolescent Substance Use and Emotional Health

    ERIC Educational Resources Information Center

    Mason, Michael J.; Korpela, Kalevi

    2009-01-01

    This study analyzed routine locations (activity spaces) of urban adolescents enrolled in a substance abuse treatment program to understand the relationship between their spatial lives and health outcomes such as substance use and mental health. Sixty-eight adolescents were interviewed and produced a list of 199 locations identified as most…

  12. Innovative Ideas for Coordinating International Space Activities: International Center for Space Medicine, International Space Authority, and other Global Youth Space Initiatives

    NASA Astrophysics Data System (ADS)

    Marshall, W.

    2002-01-01

    The Space Generation Forum SGF, at UNISPACE-III, as one of its ten formal recommendations to the United Nations in 1999, put forward the suggestion that the an international space authority should be created. Other recommendations were the establishment of an International Center for Space Medicine, creation of a global space exploration and development program, establishment of a global space (Nobel) prize, and a global space library. These projects are being further developed at the Space Generation Summit (SGS), an event at World Space Congress (WSC) which shall unite international students and young professionals to develop a youth vision and strategy for the peaceful uses of space. SGS, endorsed by the United Nations, will take place from October 11- 13th, during which the 200 delegates will discuss ongoing youth space activities, particularly those stemming from the UNISPACE-III/SGF and taken forward by the Space Generation Advisory Council. Delegates will address a variety of topics with the goal of devising new recommendations according to the theme, 'Accelerating Our Pace in Space'. The material presented here and in other technical sessions throughout WSC includes the findings of these discussions. In this paper, we present the International Space Authority idea together with recommendations on how that might be taken forward. The purpose of such an organization would be to allow: 1. Oversight and enforcement for the balanced regulation of multiple interests in space 2. Access for all peoples to the material benefits and knowledge and understanding enabled by the exploration and 3. Pooling of national and industry resources for the creation of space infrastructure, missions and enterprises for Operating principles: 1. The ISA regulatory regime would encourage commercialization and the harnessing of competitive market 2. Consistent with its charter to ensure access to all peoples, all UN member states and appropriate NGOs would 3. Close coordination with

  13. Radiation Protection Studies of International Space Station Extravehicular Activity Space Suits

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A. (Editor); Shavers, Mark R. (Editor); Saganti, Premkumar B. (Editor); Miller, Jack (Editor)

    2003-01-01

    This publication describes recent investigations that evaluate radiation shielding characteristics of NASA's and the Russian Space Agency's space suits. The introduction describes the suits and presents goals of several experiments performed with them. The first chapter provides background information about the dynamic radiation environment experienced at ISS and summarized radiation health and protection requirements for activities in low Earth orbit. Supporting studies report the development and application of a computer model of the EMU space suit and the difficulty of shielding EVA crewmembers from high-energy reentrant electrons, a previously unevaluated component of the space radiation environment. Chapters 2 through 6 describe experiments that evaluate the space suits' radiation shielding characteristics. Chapter 7 describes a study of the potential radiological health impact on EVA crewmembers of two virtually unexamined environmental sources of high-energy electrons-reentrant trapped electrons and atmospheric albedo or "splash" electrons. The radiological consequences of those sources have not been evaluated previously and, under closer scrutiny. A detailed computational model of the shielding distribution provided by components of the NASA astronauts' EMU is being developed for exposure evaluation studies. The model is introduced in Chapters 8 and 9 and used in Chapter 10 to investigate how trapped particle anisotropy impacts female organ doses during EVA. Chapter 11 presents a review of issues related to estimating skin cancer risk form space radiation. The final chapter contains conclusions about the protective qualities of the suit brought to light form these studies, as well as recommendations for future operational radiation protection.

  14. Recent Activities on the Embrace Space Weather Regional Warning Center: the New Space Weather Data Center

    NASA Astrophysics Data System (ADS)

    Denardini, Clezio Marcos; Dal Lago, Alisson; Mendes, Odim; Batista, Inez S.; SantAnna, Nilson; Gatto, Rubens; Takahashi, Hisao; Costa, D. Joaquim; Banik Padua, Marcelo; Campos Velho, Haroldo

    2016-07-01

    On August 2007 the National Institute for Space Research started a task force to develop and operate a space weather program, which is known by the acronyms Embrace that stands for the Portuguese statement "Estudo e Monitoramento BRAasileiro de Clima Espacial" Program (Brazilian Space Weather Study and Monitoring program). The mission of the Embrace/INPE program is to monitor the Solar-Terrestrial environment, the magnetosphere, the upper atmosphere and the ground induced currents to prevent effects on technological and economic activities. The Embrace/INPE system monitors the physical parameters of the Sun-Earth environment, such as Active Regions (AR) in the Sun and solar radiation by using radio telescope, Coronal Mass Ejection (CME) information by satellite and ground-based cosmic ray monitoring, geomagnetic activity by the magnetometer network, and ionospheric disturbance by ionospheric sounders and using data collected by four GPS receiver network, geomagnetic activity by a magnetometer network, and provides a forecasting for Total Electronic Content (TEC) - 24 hours ahead - using a version of the SUPIM model which assimilates the two latter data using nudging approach. Most of these physical parameters are daily published on the Brazilian space weather program web portal, related to the entire network sensors available. Regarding outreach, it has being published a daily bulletin in Portuguese and English with the status of the space weather environment on the Sun, the Interplanetary Medium and close to the Earth. Since December 2011, all these activities are carried out at the Embrace Headquarter, a building located at the INPE's main campus. Recently, a comprehensive data bank and an interface layer are under commissioning to allow an easy and direct access to all the space weather data collected by Embrace through the Embrace web Portal. The information being released encompasses data from: (a) the Embrace Digisonde Network (Embrace DigiNet) that monitors

  15. Application of CRISPR/Cas9 for biomedical discoveries.

    PubMed

    Riordan, Sean M; Heruth, Daniel P; Zhang, Li Q; Ye, Shui Qing

    2015-01-01

    The Clustered Regions of Interspersed Palindromic Repeats-Cas9 (CRISPR/Cas9), a viral defense system found in bacteria and archaea, has emerged as a tour de force genome editing tool. The CRISPR/Cas9 system is much easier to customize and optimize because the site selection for DNA cleavage is guided by a short sequence of RNA rather than an engineered protein as in the systems of zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and meganucleases. Although it still suffers from some off-target effects, the CRISPR/Cas9 system has been broadly and successfully applied for biomedical discoveries in a number of areas. In this review, we present a brief history and development of the CRISPR system and focus on the application of this genome editing technology for biomedical discoveries. We then present concise concluding remarks and future directions for this fast moving field.

  16. Application of CRISPR/Cas9 to Autophagy Research.

    PubMed

    O'Prey, J; Sakamaki, J; Baudot, A D; New, M; Van Acker, T; Tooze, S A; Long, J S; Ryan, K M

    2017-01-01

    The ability to efficiently modulate autophagy activity is paramount in the study of the field. Conventional broad-range autophagy inhibitors and genetic manipulation using RNA interference (RNAi), although widely used in autophagy research, are often limited in specificity or efficacy. In this chapter, we address the problems of conventional autophagy-modulating tools by exploring the use of three different CRISPR/Cas9 systems to abrogate autophagy in numerous human and mouse cell lines. The first system generates cell lines constitutively deleted of ATG5 or ATG7 whereas the second and third systems express a Tet-On inducible-Cas9 that enables regulated deletion of ATG5 or ATG7. We observed the efficiency of autophagy inhibition using the CRISPR/Cas9 strategy to surpass that of RNAi, and successfully generated cells with complete and sustained autophagy disruption through the CRISPR/Cas9 technology.

  17. Legal regime of human activities in outer space law

    NASA Technical Reports Server (NTRS)

    Golda, Carlo

    1994-01-01

    Current developments in space activities increasingly involve the presence of humans on board spacecraft and, in the near future, on the Moon, on Mars, on board Space Stations, etc. With respect to these challenges, the political and legal issues connected to the status of astronauts are largely unclear and require a new doctrinal attention. In the same way, many legal and political questions remain open in the structure of future space crews: the need for international standards in the definition and training of astronauts, etc.; but, first of all, an international uniform legal definition of astronauts. Moreover, the legal structure for human life and operations in outer space can be a new and relevant paradigm for the definition of similar rules in all the situations and environments in which humans are involved in extreme frontiers. The present article starts from an overview on the existing legal and political definitions of 'astronauts', moving to the search of a more useful definition. This is followed by an analysis of the concrete problems created by human space activities, and the legal and political responses to them (the need for a code of conduct; the structure of the crew and the existing rules in the US and ex-USSR; the new legal theories on the argument; the definition and structure of a code of conduct; the next legal problems in fields such as privacy law, communications law, business law, criminal law, etc.).

  18. Rational design of a split-Cas9 enzyme complex

    SciTech Connect

    Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.; Staahl, Brett T.; Bardales, Jorge A.; Kornfeld, Jack E.; Doudna, Jennifer A.

    2015-02-23

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. The lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.

  19. Rational design of a split-Cas9 enzyme complex

    DOE PAGES

    Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.; ...

    2015-02-23

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. The lobes do not interactmore » on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.« less

  20. Rational design of a split-Cas9 enzyme complex.

    PubMed

    Wright, Addison V; Sternberg, Samuel H; Taylor, David W; Staahl, Brett T; Bardales, Jorge A; Kornfeld, Jack E; Doudna, Jennifer A

    2015-03-10

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.

  1. Improving active space telescope wavefront control using predictive thermal modeling

    NASA Astrophysics Data System (ADS)

    Gersh-Range, Jessica; Perrin, Marshall D.

    2015-01-01

    Active control algorithms for space telescopes are less mature than those for large ground telescopes due to differences in the wavefront control problems. Active wavefront control for space telescopes at L2, such as the James Webb Space Telescope (JWST), requires weighing control costs against the benefits of correcting wavefront perturbations that are a predictable byproduct of the observing schedule, which is known and determined in advance. To improve the control algorithms for these telescopes, we have developed a model that calculates the temperature and wavefront evolution during a hypothetical mission, assuming the dominant wavefront perturbations are due to changes in the spacecraft attitude with respect to the sun. Using this model, we show that the wavefront can be controlled passively by introducing scheduling constraints that limit the allowable attitudes for an observation based on the observation duration and the mean telescope temperature. We also describe the implementation of a predictive controller designed to prevent the wavefront error (WFE) from exceeding a desired threshold. This controller outperforms simpler algorithms even with substantial model error, achieving a lower WFE without requiring significantly more corrections. Consequently, predictive wavefront control based on known spacecraft attitude plans is a promising approach for JWST and other future active space observatories.

  2. Integrating Multiple Space Ground Sensors to Track Volcanic Activity

    NASA Technical Reports Server (NTRS)

    Chien, Steve; Davies, Ashley; Doubleday, Joshua; Tran, Daniel; Jones, Samuel; Kjartansson, Einar; Thorsteinsson, Hrobjartur; Vogfjord, Kristin; Guomundsson, Magnus; Thordarson, Thor; Mandl, Daniel

    2011-01-01

    Volcanic activity can occur with little or no warning. Increasing numbers of space borne assets can enable coordinated measurements of volcanic events to enhance both scientific study and hazard response. We describe the use of space and ground measurements to target further measurements as part of a worldwide volcano monitoring system. We utilize a number of alert systems including the MODVOLC, GOESVOLC, US Air Force Weather Advisory, and Volcanic Ash Advisory Center (VAAC) alert systems. Additionally we use in-situ data from ground instrumentation at a number of volcanic sites, including Iceland.

  3. Benefits of advanced space suits for supporting routine extravehicular activity

    NASA Technical Reports Server (NTRS)

    Alton, L. R.; Bauer, E. H.; Patrick, J. W.

    1975-01-01

    Technology is available to produce space suits providing a quick-reaction, safe, much more mobile extravehicular activity (EVA) capability than before. Such a capability may be needed during the shuttle era because the great variety of missions and payloads complicates the development of totally automated methods of conducting operations and maintenance and resolving contingencies. Routine EVA now promises to become a cost-effective tool as less complex, serviceable, lower-cost payload designs utilizing this capability become feasible. Adoption of certain advanced space suit technologies is encouraged for reasons of economics as well as performance.

  4. Crew activity and motion effects on the space station

    NASA Technical Reports Server (NTRS)

    Rochon, Brian V.; Scheer, Steven A.

    1987-01-01

    Among the significant sources of internal disturbances that must be considered in the design of space station vibration control systems are the loads induced on the structure from various crew activities. Flight experiment T013, flown on the second manned mission of Skylab, measured force and moment time histories for a range of preplanned crew motions and activities. This experiment has proved itself invaluable as a source of on-orbit crew induced loads that has allowed a space station forcing function data base to be built. This will enable forced response such as acceleration and deflections, attributable to crew activity, to be calculated. The flight experiment, resultant database and structural model pre-processor, analysis examples and areas of combined research shall be described.

  5. A sensitive geomagnetic activity index for space weather operation

    NASA Astrophysics Data System (ADS)

    Du, D.; Xu, W. Y.; Zhao, M. X.; Chen, B.; Lu, J. Y.; Yang, G. L.

    2010-12-01

    There is an ongoing demand for real-time geomagnetic indices in space services. The traditional 3 h K index and K-derived planetary indices cannot issue alters promptly during large storms, and the 3 h interval is much larger than the time scales of ionospheric responses. To overcome these difficulties, we define a new consecutive and linear geomagnetic activity index, the range of hourly H component index (rH) with 1 min resolution, and develop a local rH index nowcast system for space weather operation, which can issue geomagnetic storm alerts quickly. We also derive Kp/Ap indices conveniently from a single station data to describe the global geomagnetic activity. Then we make a statistic comparison between rH and other definite index values during storm and find that rH is sensitive to the geomagnetic disturbance and can reflect the geomagnetic activity more delicately.

  6. INSA Scientific Activities in the Space Astronomy Area

    NASA Astrophysics Data System (ADS)

    Pérez Martínez, Ricardo; Sánchez Portal, Miguel

    Support to astronomy operations is an important and long-lived activity within INSA. Probably the best known (and traditional) INSA activities are those related with real-time spacecraft operations: ground station maintenance and operation (ground station engineers and operators); spacecraft and payload real-time operation (spacecraft and instruments controllers); computing infrastructure maintenance (operators, analysts), and general site services. In this paper, we’ll show a different perspective, probably not so well-known, presenting some INSA recent activities at the European Space Astronomy Centre (ESAC) and NASA Madrid Deep Space Communication Complex (MDSCC) directly related to scientific operations. Basic lines of activity involved include: operations support for science operations; system and software support for real time systems; technical administration and IT support; R&D activities, radioastronomy (at MDSCC and ESAC), and scientific research projects. This paper is structured as follows: first, INSA activities in two ESA cornerstone astrophysics missions, XMM-Newton and Herschel, will be outlined. Then, our activities related to scientific infrastructure services, represented by the Virtual Observatory (VO) framework and the Science Archives development facilities, are briefly shown. Radio astronomy activities will be described afterwards, and, finally, a few research topics in which INSA scientists are involved will also be described.

  7. Space activities - A review and a look ahead

    NASA Technical Reports Server (NTRS)

    Durrani, S. H.

    1984-01-01

    The paper reviews the progress made in manned and unmanned space programs during the last 25 years and names several major accomplishments. The ingredients of success are identified as good engineering, good technology, and good management of a very complex enterprise. An argument is made that the pace of progress will be governed not by technological advances, which can be very rapid, but rather by future institutional arrangements, which are much slower to evolve. It is predicted that the most likely space activities for the next 20 years will be those relating to space commercialization, and several examples are cited. A hope is expressed that policy makers and entrepreneurs will match the spirit of adventure and risk-taking exhibited by engineers in exploring uncharted territory.

  8. The interaction of beryllium with benzene and graphene: a comparative investigation based on DFT, MP2, CCSD(T), CAS-SCF and CAS-PT2.

    PubMed

    Fernandez, Nicolas; Ferro, Yves; Carissan, Yannick; Marchois, Julien; Allouche, Alain

    2014-02-07

    The interaction of beryllium with benzene, graphene and graphitic compounds involves multi-reference electronic states, Jahn-Teller distortion, charge transfer and van der Waals interactions. This is investigated herein using periodic and molecular models at different levels of theory: (i) the second-order Møller-Plesset (MP2) perturbation theory, (ii) the coupled cluster method with inclusion of single double and perturbative triple excitations (CCSD(T)), (iii) the complete active space self-consistent field (CAS-SCF) and (iv) the complete active space with perturbation theory truncated at the 2nd order (CAS-PT2). Molecular and periodic Density Functional Theory (DFT) methods are also used. The two major failures of DFT are addressed with regard to the beryllium benzene and graphene interaction: the degeneracy problem is the source of no specific problem while the delocalization error causes DFT with the Perdew Burke, Ernzerhof functional plus the Grimme correction (DFT/PBE-D2) to be over-binding by about 0.4 eV at a short-range. The agreement between DFT/PBE-D2 and wave-function based methods is nevertheless good; DFT/PBE-D2 provides an accurate description of the electronic structure of the system. By the end of this work, we shall get a better insight into the mechanisms leading beryllium to physisorb on graphene and to chemisorb into the bilayer of graphite.

  9. Modified RNAs in CRISPR/Cas9: An Old Trick Works Again.

    PubMed

    Latorre, Alfonso; Latorre, Ana; Somoza, Álvaro

    2016-03-07

    Old tricks, new dog: CRISPR/Cas9 is a powerful tool for gene editing that requires an endonuclease (Cas9) and RNA strands. It has been shown that chemical modification of the RNA structures, an approach that has been used to improve the efficiency of RNA interference, can also be applied to enhance the activity of CRISPR/Cas9 and reduce its off-target effects.

  10. The Brain in Space: A Teacher's Guide with Activities for Neuroscience.

    ERIC Educational Resources Information Center

    MacLeish, Marlene Y.; McLean, Bernice R.

    This educators guide discusses the brain and contains activities on neuroscience. Activities include: (1) "The Space Life Sciences"; (2) "Space Neuroscience: A Special Area within the Space Life Sciences"; (3) "Space Life Sciences Research"; (4) "Neurolab: A Special Space Mission to Study the Nervous System"; (5) "The Nervous System"; (6)…

  11. Potential pitfalls of CRISPR/Cas9-mediated genome editing.

    PubMed

    Peng, Rongxue; Lin, Guigao; Li, Jinming

    2016-04-01

    Recently, a novel technique named the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)9 system has been rapidly developed. This genome editing tool has improved our ability tremendously with respect to exploring the pathogenesis of diseases and correcting disease mutations, as well as phenotypes. With a short guide RNA, Cas9 can be precisely directed to target sites, and functions as an endonuclease to efficiently produce breaks in DNA double strands. Over the past 30 years, CRISPR has evolved from the 'curious sequences of unknown biological function' into a promising genome editing tool. As a result of the incessant development in the CRISPR/Cas9 system, Cas9 co-expressed with custom guide RNAs has been successfully used in a variety of cells and organisms. This genome editing technology can also be applied to synthetic biology, functional genomic screening, transcriptional modulation and gene therapy. However, although CRISPR/Cas9 has a broad range of action in science, there are several aspects that affect its efficiency and specificity, including Cas9 activity, target site selection and short guide RNA design, delivery methods, off-target effects and the incidence of homology-directed repair. In the present review, we highlight the factors that affect the utilization of CRISPR/Cas9, as well as possible strategies for handling any problems. Addressing these issues will allow us to take better advantage of this technique. In addition, we also review the history and rapid development of the CRISPR/Cas system from the time of its initial discovery in 2012.

  12. Legal considerations and cooperative opportunities for space commercial activities

    NASA Technical Reports Server (NTRS)

    Hosenball, S. N.

    1984-01-01

    It is a national policy to make the capabilities of the Space Transportation System available to a wide range of potential users. This includes its availability as a space manufacturing facility for commercial activities, which may be carried out on a reimbursable basis or as a joint endeavor with NASA, but with substantial private investment. In any high risk, long lead-time research and development activity directed towards commercialization, the protection afforded the results of the research and development under the laws relating to intellectual property rights may provide an important incentive for private investment. The policies and practices of NASA directed towards the protection of privately-established intellectual property rights involved in STS use are reviewed with particular emphasis on reimbursable launch agreements and joint endeavor agreements.

  13. Space station group activities habitability module study: A synopsis

    NASA Technical Reports Server (NTRS)

    Nixon, David; Glassman, Terry

    1987-01-01

    Space station habitability was studied by investigating crew activity routines, proximities, ergonomic envelopes, and group volumes. Ten alternative schematic interior designs were proposed. Preliminary conclusions include: (1) in-service interior modifications may be necessary and should be planned for; (2) design complexity will be increased if the module cluster is reduced from five to three; (3) the increased crew circulation attendant upon enhancement of space station activity may produce human traffic bottlenecks and should be planned for; (4) a single- or two-person quiet area may be desirable to provide crew members with needed solitude during waking hours; and (5) the decision to choose a two-shift or three-shift daily cycle will have a significant impact on the design configuration and operational efficiency of the human habitat.

  14. A 12 years brazilian space education activity experience

    NASA Astrophysics Data System (ADS)

    Stancato, Fernando; Gustavo Catalani Racca, João; Ballarotti, MaurícioG.

    2001-03-01

    A multidisciplinary group of students from the university and latter also from the high school was formed in 1988 with the objective to make them put in practice their knowledge in physics, chemistry and mathematics and engineering fields in experimental rocketry. The group was called "Grupo de Foguetes Experimentais", GFE. Since that time more than 150 students passed throw the group and now many of them are in the space arena. The benefits for students in a space hands-on project are many: More interest in their school subjects is gotten as they see an application for them; Interrelation attitudes are learned as space projects is a team activity; Responsibility is gained as each is responsible for a part of a critical mission project; Multidisciplinary and international experience is gotten as these are space project characteristics; Learn how to work in a high stress environment as use to be a project launch. This paper will cover the educational experiences gotten during these years and how some structured groups work. It is explained the objectives and how the group was formed. The group structure and the different phases that at each year the new team passes are described. It is shown the different activities that the group uses to do from scientific seminars, scientific club and international meetings to technical tours and assistance to rocket activities in regional schools. It is also explained the group outreach activities as some launches were covered by the media in more then 6 articles in newspaper and 7 television news. In 1999 as formed an official group called NATA, Núcleo de Atividades Aerospaciais within the Universidade Estadual de Londrina, UEL, by some GFE members and teachers from university. It is explained the first group project results.

  15. Active vibration control techniques for flexible space structures

    NASA Technical Reports Server (NTRS)

    Parlos, Alexander G.; Jayasuriya, Suhada

    1990-01-01

    Two proposed control system design techniques for active vibration control in flexible space structures are detailed. Control issues relevant only to flexible-body dynamics are addressed, whereas no attempt was made to integrate the flexible and rigid-body spacecraft dynamics. Both of the proposed approaches revealed encouraging results; however, further investigation of the interaction of the flexible and rigid-body dynamics is warranted.

  16. Corrosion Activities at the NASA Kennedy Space Center

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

    This report documents summer faculty fellow efforts in the corrosion test bed at the NASA Kennedy Space Center. During the summer of 2002 efforts were concentrated on three activities: a short course on corrosion control for KSC personnel, evaluation of commercial wash additives used for corrosion control on Army aircraft, and improvements in the testing of a new cathodic protection system under development at KSC.

  17. Canonical-ensemble state-averaged complete active space self-consistent field (SA-CASSCF) strategy for problems with more diabatic than adiabatic states: Charge-bond resonance in monomethine cyanines

    SciTech Connect

    Olsen, Seth

    2015-01-28

    This paper reviews basic results from a theory of the a priori classical probabilities (weights) in state-averaged complete active space self-consistent field (SA-CASSCF) models. It addresses how the classical probabilities limit the invariance of the self-consistency condition to transformations of the complete active space configuration interaction (CAS-CI) problem. Such transformations are of interest for choosing representations of the SA-CASSCF solution that are diabatic with respect to some interaction. I achieve the known result that a SA-CASSCF can be self-consistently transformed only within degenerate subspaces of the CAS-CI ensemble density matrix. For uniformly distributed (“microcanonical”) SA-CASSCF ensembles, self-consistency is invariant to any unitary CAS-CI transformation that acts locally on the ensemble support. Most SA-CASSCF applications in current literature are microcanonical. A problem with microcanonical SA-CASSCF models for problems with “more diabatic than adiabatic” states is described. The problem is that not all diabatic energies and couplings are self-consistently resolvable. A canonical-ensemble SA-CASSCF strategy is proposed to solve the problem. For canonical-ensemble SA-CASSCF, the equilibrated ensemble is a Boltzmann density matrix parametrized by its own CAS-CI Hamiltonian and a Lagrange multiplier acting as an inverse “temperature,” unrelated to the physical temperature. Like the convergence criterion for microcanonical-ensemble SA-CASSCF, the equilibration condition for canonical-ensemble SA-CASSCF is invariant to transformations that act locally on the ensemble CAS-CI density matrix. The advantage of a canonical-ensemble description is that more adiabatic states can be included in the support of the ensemble without running into convergence problems. The constraint on the dimensionality of the problem is relieved by the introduction of an energy constraint. The method is illustrated with a complete active space

  18. Canonical-ensemble state-averaged complete active space self-consistent field (SA-CASSCF) strategy for problems with more diabatic than adiabatic states: Charge-bond resonance in monomethine cyanines

    NASA Astrophysics Data System (ADS)

    Olsen, Seth

    2015-01-01

    This paper reviews basic results from a theory of the a priori classical probabilities (weights) in state-averaged complete active space self-consistent field (SA-CASSCF) models. It addresses how the classical probabilities limit the invariance of the self-consistency condition to transformations of the complete active space configuration interaction (CAS-CI) problem. Such transformations are of interest for choosing representations of the SA-CASSCF solution that are diabatic with respect to some interaction. I achieve the known result that a SA-CASSCF can be self-consistently transformed only within degenerate subspaces of the CAS-CI ensemble density matrix. For uniformly distributed ("microcanonical") SA-CASSCF ensembles, self-consistency is invariant to any unitary CAS-CI transformation that acts locally on the ensemble support. Most SA-CASSCF applications in current literature are microcanonical. A problem with microcanonical SA-CASSCF models for problems with "more diabatic than adiabatic" states is described. The problem is that not all diabatic energies and couplings are self-consistently resolvable. A canonical-ensemble SA-CASSCF strategy is proposed to solve the problem. For canonical-ensemble SA-CASSCF, the equilibrated ensemble is a Boltzmann density matrix parametrized by its own CAS-CI Hamiltonian and a Lagrange multiplier acting as an inverse "temperature," unrelated to the physical temperature. Like the convergence criterion for microcanonical-ensemble SA-CASSCF, the equilibration condition for canonical-ensemble SA-CASSCF is invariant to transformations that act locally on the ensemble CAS-CI density matrix. The advantage of a canonical-ensemble description is that more adiabatic states can be included in the support of the ensemble without running into convergence problems. The constraint on the dimensionality of the problem is relieved by the introduction of an energy constraint. The method is illustrated with a complete active space valence

  19. Canonical-ensemble state-averaged complete active space self-consistent field (SA-CASSCF) strategy for problems with more diabatic than adiabatic states: charge-bond resonance in monomethine cyanines.

    PubMed

    Olsen, Seth

    2015-01-28

    This paper reviews basic results from a theory of the a priori classical probabilities (weights) in state-averaged complete active space self-consistent field (SA-CASSCF) models. It addresses how the classical probabilities limit the invariance of the self-consistency condition to transformations of the complete active space configuration interaction (CAS-CI) problem. Such transformations are of interest for choosing representations of the SA-CASSCF solution that are diabatic with respect to some interaction. I achieve the known result that a SA-CASSCF can be self-consistently transformed only within degenerate subspaces of the CAS-CI ensemble density matrix. For uniformly distributed ("microcanonical") SA-CASSCF ensembles, self-consistency is invariant to any unitary CAS-CI transformation that acts locally on the ensemble support. Most SA-CASSCF applications in current literature are microcanonical. A problem with microcanonical SA-CASSCF models for problems with "more diabatic than adiabatic" states is described. The problem is that not all diabatic energies and couplings are self-consistently resolvable. A canonical-ensemble SA-CASSCF strategy is proposed to solve the problem. For canonical-ensemble SA-CASSCF, the equilibrated ensemble is a Boltzmann density matrix parametrized by its own CAS-CI Hamiltonian and a Lagrange multiplier acting as an inverse "temperature," unrelated to the physical temperature. Like the convergence criterion for microcanonical-ensemble SA-CASSCF, the equilibration condition for canonical-ensemble SA-CASSCF is invariant to transformations that act locally on the ensemble CAS-CI density matrix. The advantage of a canonical-ensemble description is that more adiabatic states can be included in the support of the ensemble without running into convergence problems. The constraint on the dimensionality of the problem is relieved by the introduction of an energy constraint. The method is illustrated with a complete active space valence

  20. The CRISPR/Cas9 system targeting EGFR exon 17 abrogates NF-κB activation via epigenetic modulation of UBXN1 in EGFRwt/vIII glioma cells.

    PubMed

    Huang, Kai; Yang, Chao; Wang, Qi-Xue; Li, Yan-Sheng; Fang, Chuan; Tan, Yan-Li; Wei, Jian-Wei; Wang, Yun-Fei; Li, Xin; Zhou, Jun-Hu; Zhou, Bing-Cong; Yi, Kai-Kai; Zhang, Kai-Liang; Li, Jie; Kang, Chun-Sheng

    2017-03-01

    Worldwide, glioblastoma (GBM) is the most lethal and frequent intracranial tumor. Despite decades of study, the overall survival of GBM patients remains unchanged. epidermal growth factor receptor (EGFR) amplification and gene mutation are thought to be negatively correlated with prognosis. In this study, we used proteomics to determine that UBXN1 is a negative downstream regulator of the EGFR mutation vIII (EGFRvIII). Via bioinformatics analysis, we found that UBXN1 is a factor that can improve glioma patients' overall survival time. We also determined that the down-regulation of UBXN1 is mediated by the upregulation of H3K27me3 in the presence of EGFRvIII. Because NF-κB can be negatively regulated by UBXN1, we believe that EGFRwt/vIII activates NF-κB by suppressing UBXN1 expression. Importantly, we used the latest genomic editing tool, CRISPR/Cas9, to knockout EGFRwt/vIII on exon 17 and further proved that UBXN1 is negatively regulated by EGFRwt/vIII. Furthermore, knockout of EGFR/EGFRvIII could benefit GBM in vitro and in vivo, indicating that CRISPR/Cas9 is a promising therapeutic strategy for both EGFR amplification and EGFR mutation-bearing patients.

  1. Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins.

    PubMed

    Bondy-Denomy, Joseph; Garcia, Bianca; Strum, Scott; Du, Mingjian; Rollins, MaryClare F; Hidalgo-Reyes, Yurima; Wiedenheft, Blake; Maxwell, Karen L; Davidson, Alan R

    2015-10-01

    The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR-Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR-Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. In vivo, this anti-CRISPR can convert the CRISPR-Cas system into a transcriptional repressor, providing the first example-to our knowledge-of modulation of CRISPR-Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR-Cas function.

  2. NASA Stennis Space Center Test Technology Branch Activities

    NASA Technical Reports Server (NTRS)

    Solano, Wanda M.

    2000-01-01

    This paper provides a short history of NASA Stennis Space Center's Test Technology Laboratory and briefly describes the variety of engine test technology activities and developmental project initiatives. Theoretical rocket exhaust plume modeling, acoustic monitoring and analysis, hand held fire imaging, heat flux radiometry, thermal imaging and exhaust plume spectroscopy are all examples of current and past test activities that are briefly described. In addition, recent efforts and visions focused on accomodating second, third, and fourth generation flight vehicle engine test requirements are discussed.

  3. The LATT way towards large active primaries for space telescopes

    NASA Astrophysics Data System (ADS)

    Briguglio, Runa; Arcidiacono, Carmelo; Xompero, Marco; Lisi, Franco; Riccardi, Armando; Biasi, Roberto; Patauner, Christian; Gallieni, Daniele; Lazzarini, Paolo; Tintori, Matteo; d'Amato, Francesco; Pucci, Mauro; Duò, Fabrizio; Vettore, Christian; Zuccaro Marchi, Alessandro

    2016-07-01

    The Large Aperture Telescope Technology (LATT) goes beyond the current paradigm of future space telescopes, based on a deformable mirror in the pupil relay. Through the LATT project we demonstrated the concept of a low-weight active primary mirror, whose working principle and control strategy benefit from two decades of advances in adaptive optics for ground-based telescopes. We developed a forty centimeter spherical mirror prototype, with an areal density lower than 17 kg/m2, controlled through contactless voice coil actuators with co-located capacitive position sensors. The prototype was subjected to thermo-vacuum, vibration and optical tests, to push its technical readiness toward level 5. In this paper we present the background and the outcomes of the LATT activities under ESA contract (TRP programme), exploring the concept of a lightweight active primary mirror for space telescopes. Active primaries will open the way to very large segmented apertures, actively shaped, which can be lightweight, deployable and accurately phased once in flight.

  4. 48 CFR 1852.228-76 - Cross-waiver of liability for space station activities.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... for space station activities. 1852.228-76 Section 1852.228-76 Federal Acquisition Regulations System... CLAUSES Texts of Provisions and Clauses 1852.228-76 Cross-waiver of liability for space station activities... Space Station Activities (DEC 1994) (a) The Intergovernmental Agreement for the Space Station contains...

  5. A New Active Space Radiation Instruments for the International Space Station, A-DREAMS

    NASA Astrophysics Data System (ADS)

    Uchihori, Yukio; Kodaira, Satoshi; Kitamura, Hisashi; Kobayashi, Shingo

    For future space experiments in the International Space Station (ISS) or other satellites, radiation detectors, A-DREAMS (Active Dosimeter for Radiation Environment and Astronautic Monitoring in Space), using single or multiple silicon semi-conductor detectors have been developed. The first version of the detectors were produced and calibrated with particle accelerators. National Institute of Radiological Sciences has a medical heavy ion accelerator (HIMAC) for cancer therapy and a cyclotron accelerator. The detector was irradiated with high energy heavy ions and protons in HIMAC and the cyclotron and calibrated the energy resolution and linearity for deposited energies of these particles. We are planned to be going to use the new instrument in an international project, the new MATROSHKA experiment which is directed by members in the Institute of Bio-Medical Problem (IBMP) in Russia and German Space Center (DLR) in Germany. In the project, the dose distribution in human torso phantom will be investigated for several months in the ISS. For the project, a new type of the instruments is under development in NIRS and the current situation will be reported in this paper.

  6. Active and passive vibration suppression for space structures

    NASA Technical Reports Server (NTRS)

    Hyland, David C.

    1991-01-01

    The relative benefits of passive and active vibration suppression for large space structures (LSS) are discussed. The intent is to sketch the true ranges of applicability of these approaches using previously published technical results. It was found that the distinction between active and passive vibration suppression approaches is not as sharp as might be thought at first. The relative simplicity, reliability, and cost effectiveness touted for passive measures are vitiated by 'hidden costs' bound up with detailed engineering implementation issues and inherent performance limitations. At the same time, reliability and robustness issues are often cited against active control. It is argued that a continuum of vibration suppression measures offering mutually supporting capabilities is needed. The challenge is to properly orchestrate a spectrum of methods to reap the synergistic benefits of combined advanced materials, passive damping, and active control.

  7. Progress of application and off-target effects of CRISPR/Cas9.

    PubMed

    Wu, Zheng; Feng, Gu

    2015-10-01

    The clustered regulatory interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system mediates genome editing and is revolutionizing genetic researches. Scientists are able to manipulate the gene of interest from any organism with CRISPR/Cas9. Compared with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) technologies, the CRISPR/Cas9 technology provides an easy and efficient approach to manipulate the genome. In this system, sgRNA (Single guide RNA), a short RNA matching the targeted DNA fragment, guides the CRISPR/Cas9 to interrogate the genome. Because sgRNA can tolerate certain mismatches to the DNA targets and thereby promote undesired off-target mutagenesis, the key limit of this technology is off-target effects. To eliminate the off-target effects, different strategies have been adopted. In this review, we summarize the application of CRISPR/Cas9 and different strategies for addressing off-target effects.

  8. Integration and exchange of split dCas9 domains for transcriptional controls in mammalian cells

    PubMed Central

    Ma, Dacheng; Peng, Shuguang; Xie, Zhen

    2016-01-01

    Programmable and precise regulation of dCas9 functions in response to multiple molecular signals by using synthetic gene circuits will expand the application of the CRISPR-Cas technology. However, the application of CRISPR-Cas therapeutic circuits is still challenging due to the restrictive cargo size of existing viral delivery vehicles. Here, we construct logic AND circuits by integrating multiple split dCas9 domains, which is useful to reduce the size of synthetic circuits. In addition, we engineer sensory switches by exchanging split dCas9 domains, allowing differential regulations on one gene, or activating two different genes in response to cell-type specific microRNAs. Therefore, we provide a valuable split-dCas9 toolkit to engineer complex transcription controls, which may inspire new biomedical applications. PMID:27694915

  9. Understanding human activity patterns based on space-time-semantics

    NASA Astrophysics Data System (ADS)

    Huang, Wei; Li, Songnian

    2016-11-01

    Understanding human activity patterns plays a key role in various applications in an urban environment, such as transportation planning and traffic forecasting, urban planning, public health and safety, and emergency response. Most existing studies in modeling human activity patterns mainly focus on spatiotemporal dimensions, which lacks consideration of underlying semantic context. In fact, what people do and discuss at some places, inferring what is happening at the places, cannot be simple neglected because it is the root of human mobility patterns. We believe that the geo-tagged semantic context, representing what individuals do and discuss at a place and a specific time, drives a formation of specific human activity pattern. In this paper, we aim to model human activity patterns not only based on space and time but also with consideration of associated semantics, and attempt to prove a hypothesis that similar mobility patterns may have different motivations. We develop a spatiotemporal-semantic model to quantitatively express human activity patterns based on topic models, leading to an analysis of space, time and semantics. A case study is conducted using Twitter data in Toronto based on our model. Through computing the similarities between users in terms of spatiotemporal pattern, semantic pattern and spatiotemporal-semantic pattern, we find that only a small number of users (2.72%) have very similar activity patterns, while the majority (87.14%) show different activity patterns (i.e., similar spatiotemporal patterns and different semantic patterns, similar semantic patterns and different spatiotemporal patterns, or different in both). The population of users that has very similar activity patterns is decreased by 56.41% after incorporating semantic information in the corresponding spatiotemporal patterns, which can quantitatively prove the hypothesis.

  10. NASA Aerosciences Activities to Support Human Space Flight

    NASA Technical Reports Server (NTRS)

    LeBeau, Gerald J.

    2011-01-01

    The Lyndon B. Johnson Space Center (JSC) has been a critical element of the United State's human space flight program for over 50 years. It is the home to NASA s Mission Control Center, the astronaut corps, and many major programs and projects including the Space Shuttle Program, International Space Station Program, and the Orion Project. As part of JSC's Engineering Directorate, the Applied Aeroscience and Computational Fluid Dynamics Branch is charted to provide aerosciences support to all human spacecraft designs and missions for all phases of flight, including ascent, exo-atmospheric, and entry. The presentation will review past and current aeroscience applications and how NASA works to apply a balanced philosophy that leverages ground testing, computational modeling and simulation, and flight testing, to develop and validate related products. The speaker will address associated aspects of aerodynamics, aerothermodynamics, rarefied gas dynamics, and decelerator systems, involving both spacecraft vehicle design and analysis, and operational mission support. From these examples some of NASA leading aerosciences challenges will be identified. These challenges will be used to provide foundational motivation for the development of specific advanced modeling and simulation capabilities, and will also be used to highlight how development activities are increasing becoming more aligned with flight projects. NASA s efforts to apply principles of innovation and inclusion towards improving its ability to support the myriad of vehicle design and operational challenges will also be briefly reviewed.

  11. SD-CAS: Spin Dynamics by Computer Algebra System.

    PubMed

    Filip, Xenia; Filip, Claudiu

    2010-11-01

    A computer algebra tool for describing the Liouville-space quantum evolution of nuclear 1/2-spins is introduced and implemented within a computational framework named Spin Dynamics by Computer Algebra System (SD-CAS). A distinctive feature compared with numerical and previous computer algebra approaches to solving spin dynamics problems results from the fact that no matrix representation for spin operators is used in SD-CAS, which determines a full symbolic character to the performed computations. Spin correlations are stored in SD-CAS as four-entry nested lists of which size increases linearly with the number of spins into the system and are easily mapped into analytical expressions in terms of spin operator products. For the so defined SD-CAS spin correlations a set of specialized functions and procedures is introduced that are essential for implementing basic spin algebra operations, such as the spin operator products, commutators, and scalar products. They provide results in an abstract algebraic form: specific procedures to quantitatively evaluate such symbolic expressions with respect to the involved spin interaction parameters and experimental conditions are also discussed. Although the main focus in the present work is on laying the foundation for spin dynamics symbolic computation in NMR based on a non-matrix formalism, practical aspects are also considered throughout the theoretical development process. In particular, specific SD-CAS routines have been implemented using the YACAS computer algebra package (http://yacas.sourceforge.net), and their functionality was demonstrated on a few illustrative examples.

  12. Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes

    PubMed Central

    Fine, Eli J.; Appleton, Caleb M.; White, Douglas E.; Brown, Matthew T.; Deshmukh, Harshavardhan; Kemp, Melissa L.; Bao, Gang

    2015-01-01

    CRISPR/Cas9 systems have been used in a wide variety of biological studies; however, the large size of CRISPR/Cas9 presents challenges in packaging it within adeno-associated viruses (AAVs) for clinical applications. We identified a two-cassette system expressing pieces of the S. pyogenes Cas9 (SpCas9) protein which splice together in cellula to form a functional protein capable of site-specific DNA cleavage. With specific CRISPR guide strands, we demonstrated the efficacy of this system in cleaving the HBB and CCR5 genes in human HEK-293T cells as a single Cas9 and as a pair of Cas9 nickases. The trans-spliced SpCas9 (tsSpCas9) displayed ~35% of the nuclease activity compared with the wild-type SpCas9 (wtSpCas9) at standard transfection doses, but had substantially decreased activity at lower dosing levels. The greatly reduced open reading frame length of the tsSpCas9 relative to wtSpCas9 potentially allows for more complex and longer genetic elements to be packaged into an AAV vector including tissue-specific promoters, multiplexed guide RNA expression, and effector domain fusions to SpCas9. For unknown reasons, the tsSpCas9 system did not work in all cell types tested. The use of protein trans-splicing may help facilitate exciting new avenues of research and therapeutic applications through AAV-based delivery of CRISPR/Cas9 systems. PMID:26126518

  13. Carbon Nanotube Activities at NASA-Johnson Space Center

    NASA Technical Reports Server (NTRS)

    Arepalli, Sivaram

    2006-01-01

    Research activities on carbon nanotubes at NASA-Johnson Space Center include production, purification, characterization and their applications for human space flight. In-situ diagnostics during nanotube production by laser oven process include collection of spatial and temporal data of passive emission and laser induced fluorescence from C2, C3 and Nickel atoms in the plume. Details of the results from the "parametric study" of the pulsed laser ablation process indicate the effect of production parameters including temperature, buffer gas, flow rate, pressure, and laser fluence. Improvement of the purity by a variety of steps in the purification process is monitored by characterization techniques including SEM, TEM, Raman, UV-VIS-NIR and TGA. A recently established NASA-JSC protocol for SWCNT characterization is undergoing revision with feedback from nanotube community. Efforts at JSC over the past five years in composites have centered on structural polymednanotube systems. Recent activities broadened this focus to multifunctional materials, supercapacitors, fuel cells, regenerable CO2 absorbers, electromagnetic shielding, radiation dosimetry and thermal management systems of interest for human space flight. Preliminary tests indicate improvement of performance in most of these applications because of the large surface area as well as high electrical and thermal conductivity exhibited by SWCNTs.

  14. Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes.

    PubMed

    Ma, Enbo; Harrington, Lucas B; O'Connell, Mitchell R; Zhou, Kaihong; Doudna, Jennifer A

    2015-11-05

    Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9-guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family.

  15. Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

    PubMed Central

    Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

    2015-01-01

    Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

  16. The Active Space of Mexican Rice Borer Pheromone Traps.

    PubMed

    Wilson, Blake E; Beuzelin, Julien M; Allison, Jeremy D; Reagan, Thomas E

    2016-09-01

    The Mexican rice borer, Eoreuma loftini (Dyar) (Lepidoptera: Crambidae), is an invasive pest of sugarcane, Saccharum spp., rice, Oryza sativa L., and other graminaceous crops in the United States. Traps baited with the synthetic female sex pheromone of E. loftini are used for monitoring and management of this invasive pest. However, the active space, or radius of attraction, of these traps is not known. Two field experiments examined the effect of intertrap distance on trap captures with hexagonal arrays of traps deployed in rice stubble habitat in Texas (2011) and Louisiana (2013). Trap capture increased with increasing intertrap distance. Trap interference occurred at intertrap distances ≤50 m in the 2011 experiment. Results from the experiment conducted in 2013 indicate that trap interference occurs at intertrap distances of 50 m, but not at distances ≥100 m. These results suggest that under field conditions, E. loftini pheromone traps attract males from distances of 50-100 m. The active space of pheromone traps also was examined under controlled wind conditions by direct observation of male response to detection of the female sex pheromone. Eoreuma loftini males responded to the pheromone blend by becoming active, fanning their wings, and rapidly walking in circles. The mean distance from the pheromone source at which males responded was 47.6 m. This work provides the first documentation of active space for traps baited with female sex pheromone for a crambid species, and these data will improve pheromone trap deployment strategies for E. loftini monitoring and management.

  17. The radii of SU Cas and TU Cas

    NASA Technical Reports Server (NTRS)

    Niva, G. D.; Schmidt, E. G.

    1980-01-01

    It is possible to obtain the masses of Cepheid variables by several methods involving the pulsation theory. However, these masses are frequently smaller than those indicated by the theory of stellar evolution. The cause of this discrepancy is not fully understood. Since the pulsation theory indicates that there is a relation among the mass, the radius and the period, the discrepancy also manifests itself in the radii of these stars. With this in mind, radius determinations for two Cepheids, SU Cas and TU Cas, were undertaken. It is concluded that because of the agreement between the present radius and the beat radius of TU Cas, the pulsation theory is giving correct information about the radii of beat Cepheids. This implies that the luminosities of short period Cepheids have been overestimated. Thus, the solution to the mass discrepancy should perhaps be sought in the theory of stellar evolution or in the possibility of mass loss.

  18. Spacing and strength of active continental strike-slip faults

    NASA Astrophysics Data System (ADS)

    Zuza, Andrew V.; Yin, An; Lin, Jessica; Sun, Ming

    2017-01-01

    Parallel and evenly-spaced active strike-slip faults occur widely in nature across diverse tectonic settings. Despite their common existence, the fundamental question of what controls fault spacing remains unanswered. Here we present a mechanical model for the generation of parallel strike-slip faults that relates fault spacing to the following parameters: (1) brittle-crust thickness, (2) fault strength, (3) crustal strength, and (4) crustal stress state. Scaled analogue experiments using dry sand, dry crushed walnut shells, and viscous putty were employed to test the key assumptions of our quantitative model. The physical models demonstrate that fault spacing (S) is linearly proportional to brittle-layer thickness (h), both in experiments with only brittle materials and in two-layer trials involving dry sand overlying viscous putty. The S / h slope in the two-layer sand-putty experiments may be controlled by the (1) rheological/geometric properties of the viscous layer, (2) effects of distributed basal loading caused by the viscous shear of the putty layer, and/or (3) frictional interaction at the sand-putty interface (i.e., coupling between the viscous and brittle layers). We tentatively suggest that this third effect exerts the strongest control on fault spacing in the analogue experiments. By applying our quantitative model to crustal-scale strike-slip faults using fault spacing and the seismogenic-zone thickness obtained from high-resolution earthquake-location data, we estimate absolute fault friction of active strike-slip faults in Asia and along the San Andreas fault system in California. We show that the average friction coefficient of strike-slip faults in the India-Asia collisional orogen is lower than that of faults in the San Andreas fault system. Weaker faults explain why deformation penetrates >3500 km into Asia from the Himalaya and why the interior of Asia is prone to large (M > 7.0) devastating earthquakes along major intra-continental strike

  19. Physical, antioxidant and structural characterization of blend films based on hsian-tsao gum (HG) and casein (CAS).

    PubMed

    Yang, Hui; Wen, Xiao Long; Guo, Shan Guang; Chen, Ming Tsao; Jiang, Ai Min; Lai, Lih-Shiuh

    2015-12-10

    The effects of hsian-tsao gum (HG) addition on the physical properties, antioxidant activities and structure of casein (CAS) film have been investigated. It has been observed that HG addition provided CAS film with better mechanical properties and resistant to moisture, stronger barrier properties against light and higher antioxidant activities than pure CAS film. Fourier transformation infrared (FTIR) data indicated that hydrogen bonding interactions and Maillard reactions occurred between CAS and HG, giving rise to a more compact structure than CAS film. The results of X-ray diffraction and differential scanning calorimetry (DSC) indicated that CAS and HG were compatible, and addition of HG destroyed the original crystalline domains of CAS film, and the blend films exhibited higher glass transition temperatures than CAS film. Moreover, nuclear magnetic resonance (NMR) analysis showed that HG addition significantly changed the mobility of water molecule in CAS film. Especially, ratio of the high mobility water of CAS/HG films significantly decreased as compared to CAS film.

  20. Geometry optimization of radicaloid systems using improved virtual orbital-complete active space configuration interaction (IVO-CASCI) analytical gradient method.

    PubMed

    Chattopadhyay, Sudip; Chaudhuri, Rajat K; Freed, Karl F

    2011-04-28

    The improved virtual orbital (IVO) complete active space (CAS) configuration interaction (IVO-CASCI) method is a simplified CAS self-consistent field (SCF), CASSCF, method. Unlike the CASSCF approach, the IVO-CASCI method does not require iterations beyond an initial SCF calculation, rendering the IVO-CASCI scheme computationally more tractable than the CASSCF method and devoid of the convergence problems that sometimes plague CASSCF calculations as the CAS size increases, while retaining all the essential positive benefits of the CASSCF method. Earlier applications demonstrate that the IVO-CASCI energies are at least as accurate as those from the CASSCF and provide the impetus for our recent development of the analytical derivative procedures that are necessary for a wide applicability of the IVO-CASCI approach. Here we test the ability of the analytic energy gradient IVO-CASCI approach (which can treat both closed- and open-shell molecules of arbitrary spin multiplicity) to compute the equilibrium geometries of four organic radicaloid species, namely, (i) the diradicals trimethylenemethane (TMM), 2,6-pyridyne, and the 2,6-pyridynium cation and (ii) a triradical 1,2,3-tridehydrobenzene (TDB), using various basis sets and different choices for the active space. Although these systems and related molecules have fascinated theoretical chemists for many years, their strong multireference character makes their description quite difficult with most standard many-body approaches. Thus, they provide ideal tests to assess the performance of the IVO-CASCI method. The present work demonstrates consistent agreement with far more expensive benchmark state-of-the-art ab initio calculations and thereby indicates that this new gradient method is able to describe the geometries of various radicaloids very accurately, even when small, but qualitatively correct, reference spaces are used. For example, the IVO-CASCI method leads to a monocyclic structure for the 2,6-isomers of the

  1. Space Power Amplification with Active Linearly Tapered Slot Antenna Array

    NASA Technical Reports Server (NTRS)

    Simons, Rainee N.; Lee, Richard Q.

    1993-01-01

    A space power amplifier composed of active linearly tapered slot antennas (LTSA's) has been demonstrated and shown to have a gain of 30 dB at 20 GHz. In each of the antenna elements, a GaAs monolithic microwave integrated circuit (MMIC) three-stage power amplifier is integrated with two LTSA's. The LTSA and the MMIC power amplifier has a gain of 11 dB and power added efficiency of 14 percent respectively. The design is suitable for constructing a large array using monolithic integration techniques.

  2. Searching the Future for the Legal Regime of Space Activities: the Need for Unification of National Space Legislation' Provisions

    NASA Astrophysics Data System (ADS)

    Negoda, S. A.

    2002-01-01

    space activities. For the future legal regime of space activities it is vital to preserve the existed principles and main provisions of the international space law. related legislations are developing rapidly. They become serious instrument for legal regulation of space activities. those projects with a foreign party involvement. Quite often partners in international space projects agree to choice a domestic law of one of them. They do this for defining a certain organizational and/or contractual issue (disputes settlement, for example) of the project. that such practice will spread widely. could help to preserve the existed important provisions of international space law (responsibility of states for their national activities, for instance). development of international space private law. We believe that solely special laws and regulations of national legislations could not regulate modern space activities. Being more and more commercial, space activities are becoming a real part of "downed to Earth" commercial activities. Therefore, in many countries provisions of civil, commercial, investment and other branches of national law are applied to such activities. which could low possible risks of such activities and to control them. Such unification seems to be suitable in the following fields: 1)implementation of provisions of international space law in national space laws; 2)definition of unified terminology, accepted by national laws of all parties; 3)unification in national legislations of a certain standards (insurance rates and rules, for instance); 4)unification in national laws of issues related to liability (for instance, a mutual wave of liability in certain types of 5)implementation in national laws of unified rules and procedures of space-related commercial disputes settlement; 6)unification of mechanisms for protection of space-related intellectual property. unification of their provisions. Special attention is paid to provisions of private law

  3. crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus.

    PubMed

    Karvelis, Tautvydas; Gasiunas, Giedrius; Miksys, Algirdas; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus

    2013-05-01

    The Cas9-crRNA complex of the Streptococcus thermophilus DGCC7710 CRISPR3-Cas system functions as an RNA-guided endonuclease with crRNA-directed target sequence recognition and protein-mediated DNA cleavage. We show here that an additional RNA molecule, tracrRNA (trans-activating CRISPR RNA), co-purifies with the Cas9 protein isolated from the heterologous E. coli strain carrying the S. thermophilus DGCC7710 CRISPR3-Cas system. We provide experimental evidence that tracrRNA is required for Cas9-mediated DNA interference both in vitro and in vivo. We show that Cas9 specifically promotes duplex formation between the precursor crRNA (pre-crRNA) transcript and tracrRNA, in vitro. Furthermore, the housekeeping RNase III contributes to primary pre-crRNA-tracrRNA duplex cleavage for mature crRNA biogenesis. RNase III, however, is not required in the processing of a short pre-crRNA transcribed from a minimal CRISPR array containing a single spacer. Finally, we show that an in vitro-assembled ternary Cas9-crRNA-tracrRNA complex cleaves DNA. This study further specifies the molecular basis for crRNA-based re-programming of Cas9 to specifically cleave any target DNA sequence for precise genome surgery. The processes for crRNA maturation and effector complex assembly established here will contribute to the further development of the Cas9 re-programmable system for genome editing applications.

  4. Gene transcription repression in Clostridium beijerinckii using CRISPR-dCas9.

    PubMed

    Wang, Yi; Zhang, Zhong-Tian; Seo, Seung-Oh; Lynn, Patrick; Lu, Ting; Jin, Yong-Su; Blaschek, Hans P

    2016-12-01

    CRISPR-Cas9 has been explored as a powerful tool for genome engineering for many organisms. Meanwhile, dCas9 which lacks endonuclease activity but can still bind to target loci has been engineered for efficient gene transcription repression. Clostridium beijerinckii, an industrially significant species capable of biosolvent production, is generally difficult to metabolically engineer. Recently, we reported our work in developing customized CRISPR-Cas9 system for genome engineering in C. beijerinckii. However, in many cases, gene expression repression (rather than actual DNA mutation) is more desirable for various biotechnological applications. Here, we further demonstrated gene transcription repression in C. beijerinckii using CRISPR-dCas9. A small RNA promoter was employed to drive the expression of the single chimeric guide RNA targeting on the promoter region of amylase gene, while a constitutive thiolase promoter was used to drive Streptococcus pyogenes dCas9 expression. The growth assay on starch agar plates showed qualitatively significant repression of amylase activity in C. beijerinckii transformant with CRISPR-dCas9 compared to the control strain. Further amylase activity quantification demonstrated consistent repression (65-97% through the fermentation process) on the activity in the transformant with CRISPR-dCas9 versus in the control. Our results provided essential references for engineering CRISPR-dCas9 as an effective tool for tunable gene transcription repression in diverse microorganisms. Biotechnol. Bioeng. 2016;113: 2739-2743. © 2016 Wiley Periodicals, Inc.

  5. Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

    PubMed Central

    Aouida, Mustapha; Eid, Ayman; Ali, Zahir; Cradick, Thomas; Lee, Ciaran; Deshmukh, Harshavardhan; Atef, Ahmed; AbuSamra, Dina; Gadhoum, Samah Zeineb; Merzaban, Jasmeen; Bao, Gang; Mahfouz, Magdy

    2015-01-01

    The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells. PMID:26225561

  6. p130Cas over-expression impairs mammary branching morphogenesis in response to estrogen and EGF.

    PubMed

    Camacho Leal, Maria del Pilar; Pincini, Alessandra; Tornillo, Giusy; Fiorito, Elisa; Bisaro, Brigitte; Di Luca, Elisa; Turco, Emilia; Defilippi, Paola; Cabodi, Sara

    2012-01-01

    p130Cas adaptor protein regulates basic processes such as cell cycle control, survival and migration. p130Cas over-expression has been related to mammary gland transformation, however the in vivo consequences of p130Cas over-expression during mammary gland morphogenesis are not known. In ex vivo mammary explants from MMTV-p130Cas transgenic mice, we show that p130Cas impairs the functional interplay between Epidermal Growth Factor Receptor (EGFR) and Estrogen Receptor (ER) during mammary gland development. Indeed, we demonstrate that p130Cas over-expression upon the concomitant stimulation with EGF and estrogen (E2) severely impairs mammary morphogenesis giving rise to enlarged multicellular spherical structures with altered architecture and absence of the central lumen. These filled acinar structures are characterized by increased cell survival and proliferation and by a strong activation of Erk1/2 MAPKs and Akt. Interestingly, antagonizing the ER activity is sufficient to re-establish branching morphogenesis and normal Erk1/2 MAPK activity. Overall, these results indicate that high levels of p130Cas expression profoundly affect mammary morphogenesis by altering epithelial architecture, survival and unbalancing Erk1/2 MAPKs activation in response to growth factors and hormones. These results suggest that alteration of morphogenetic pathways due to p130Cas over-expression might prime mammary epithelium to tumorigenesis.

  7. Attitude and CAS Use in Senior Secondary Mathematics: A Case Study of Seven Year 11 Students

    ERIC Educational Resources Information Center

    Cameron, Scott; Ball, Lynda

    2014-01-01

    This paper investigates the possible influence of attitude on seven Year 11 students' use of a Computer Algebra System (CAS) during a class activity where students could choose to use CAS or pen-and-paper in solving a range of problems. Investigation of anxiety, confidence, liking and usefulness through a survey and interview revealed that these…

  8. Space Suit CO2 Washout During Intravehicular Activity

    NASA Technical Reports Server (NTRS)

    Augustine, Phillip M.; Navarro, Moses; Conger, Bruce; Sargusingh, Miriam M.

    2010-01-01

    Space suit carbon dioxide (CO2) washout refers to the removal of CO2 gas from the oral-nasal area of a suited astronaut's (or crewmember's) helmet using the suit's ventilation system. Inadequate washout of gases can result in diminished mental/cognitive abilities as well as headaches and light headedness. In addition to general discomfort, these ailments can impair an astronaut s ability to perform mission-critical tasks ranging from flying the space vehicle to performing lunar extravehicular activities (EVAs). During design development for NASA s Constellation Program (CxP), conflicting requirements arose between the volume of air flow that the new Orion manned space vehicle is allocated to provide to the suited crewmember and the amount of air required to achieve CO2 washout in a space suit. Historically, space suits receive 6.0 actual cubic feet per minute (acfm) of air flow, which has adequately washed out CO2 for EVAs. For CxP, the Orion vehicle will provide 4.5 acfm of air flow to the suit. A group of subject matter experts (SM Es) among the EVA Systems community came to an early consensus that 4.5 acfm may be acceptable for low metabolic rate activities. However, this value appears very risky for high metabolic rates, hence the need for further analysis and testing. An analysis was performed to validate the 4.5 acfm value and to determine if adequate CO2 washout can be achieved with the new suit helmet design concepts. The analysis included computational fluid dynamic (CFD) modeling cases, which modeled the air flow and breathing characteristics of a human wearing suit helmets. Helmet testing was performed at the National Institute of Occupational Safety and Health (NIOSH) in Pittsburgh, Pennsylvania, to provide a gross-level validation of the CFD models. Although there was not a direct data correlation between the helmet testing and the CFD modeling, the testing data showed trends that are very similar to the CFD modeling. Overall, the analysis yielded

  9. Plasma Hazards and Acceptance for International Space Station Extravehicular Activities

    NASA Astrophysics Data System (ADS)

    Patton, Thomas

    2010-09-01

    Extravehicular activity(EVA) is accepted by NASA and other space faring agencies as a necessary risk in order to build and maintain a safe and efficient laboratory in space. EVAs are used for standard construction and as contingency operations to repair critical equipment for vehicle sustainability and safety of the entire crew in the habitable volume. There are many hazards that are assessed for even the most mundane EVA for astronauts, and the vast majority of these are adequately controlled per the rules of the International Space Station Program. The need for EVA repair and construction has driven acceptance of a possible catastrophic hazard to the EVA crewmember which cannot currently be controlled adequately. That hazard is electrical shock from the very environment in which they work. This paper describes the environment, causes and contributors to the shock of EVA crewmembers attributed to the ionospheric plasma environment in low Earth orbit. It will detail the hazard history, and acceptance process for the risk associated with these hazards that give assurance to a safe EVA. In addition to the hazard acceptance process this paper will explore other factors that go into the decision to accept a risk including criticality of task, hardware design and capability, and the probability of hazard occurrence. Also included will be the required interaction between organizations at NASA(EVA Office, Environments, Engineering, Mission Operations, Safety) in order to build and eventually gain adequate acceptance rationale for a hazard of this kind. During the course of the discussion, all current methods of mitigating the hazard will be identified. This paper will capture the history of the plasma hazard analysis and processes used by the International Space Station Program to formally assess and qualify the risk. The paper will discuss steps that have been taken to identify and perform required analysis of the floating potential shock hazard from the ISS environment

  10. Active Magnetic Shielding for Long Duration Manned Space Missions

    NASA Astrophysics Data System (ADS)

    Battiston, R.; Burger, W. J.; Cavelli, V.; Musenich, R.; Datskov, V. I.; Della Torre, A.; Venditti, F.; Hovland, S.; Meinke, R. B.; Van Sciver, S.; Westover, S. C.; Spillantini, P.

    2013-09-01

    The radiation risk due to ionizing particles is a critical issue for long duration manned space missions. The ionization losses in the materials of the spacecraft provide passive shielding effectively stopping low energy particles. However, the estimates of the material required to obtain an acceptable level of radiation result in a prohibitive mass. Active electromagnetic shields, which deflect the charged particles, have been considered as an alternative solution. A study of active magnetic shielding based on high-temperature superconductors (HTS) was initiated in an ESA study, and continued in the context of the NASA Innovative Advanced Concepts (NIAC) program. The aim of the effort was to provide a realistic evaluation of the possibilities based on the current technological level. The different configurations considered were assessed in terms of their technical feasibility and shielding efficiency.

  11. Active illuminated space object imaging and tracking simulation

    NASA Astrophysics Data System (ADS)

    Yue, Yufang; Xie, Xiaogang; Luo, Wen; Zhang, Feizhou; An, Jianzhu

    2016-10-01

    Optical earth imaging simulation of a space target in orbit and it's extraction in laser illumination condition were discussed. Based on the orbit and corresponding attitude of a satellite, its 3D imaging rendering was built. General simulation platform was researched, which was adaptive to variable 3D satellite models and relative position relationships between satellite and earth detector system. Unified parallel projection technology was proposed in this paper. Furthermore, we denoted that random optical distribution in laser-illuminated condition was a challenge for object discrimination. Great randomicity of laser active illuminating speckles was the primary factor. The conjunction effects of multi-frame accumulation process and some tracking methods such as Meanshift tracking, contour poid, and filter deconvolution were simulated. Comparison of results illustrates that the union of multi-frame accumulation and contour poid was recommendable for laser active illuminated images, which had capacities of high tracking precise and stability for multiple object attitudes.

  12. New CRISPR-Cas systems discovered.

    PubMed

    Yang, Hui; Patel, Dinshaw J

    2017-03-01

    In bacteria and archaea, CRISPR-Cas adaptive immune systems utilize RNA-guided endonucleases to defend against invasion by foreign nucleic acids of bacteriophage, virus and plasmid origin. In a recent paper published in Nature, Burstein et al. identified the first Cas9 protein in uncultivated archaea and two novel CRISPR-CasX and CRISPR-CasY systems in uncultivated bacteria by capitalizing on analysis of terabase-scale metagenomic datasets from natural uncultivated organisms.

  13. Nanotube Activities at NASA-Johnson Space Center

    NASA Technical Reports Server (NTRS)

    Arepalli, Sivaram

    2004-01-01

    Nanotube activities at NASA-Johnson Space Center include production, purification, characterization as well as applications of single wall carbon nanotubes (SWCNTs). A parametric study of the pulsed laser ablation process is recently completed to monitor the effect of production parameters including temperature, buffer gas, flow rate, pressure, and laser fluence. Enhancement of production is achieved by rastering the graphite target and by increasing the target surface temperature with a cw laser. In-situ diagnostics during production included time resolved passive emission and laser induced fluorescence from the plume. The improvement of the purity by a variety of steps in the purification process is monitored by characterization techniques including SEM, TEM, Raman, UV-VIS-NIR and TGA. A recently established NASA-JSC protocol for SWCNT characterization is undergoing revision with feedback from nanotube community. Efforts at JSC over the past five years in composites have centered on structural polymer/nanotube systems. Recent activities broadened this focus to multifunctional materials, supercapacitors, fuel cells, regenerable CO2 absorbers, electromagnetic shielding, radiation dosimetry and thermal management systems of interest for human space flight. Preliminary tests indicate improvement of performance in most of these applications because of the large Surface area as well as high electrical and thermal conductivity exhibited by SWCNTs. Comparison with existing technologies and possible future improvements in the SWCNT materials sill be presented.

  14. 48 CFR 1828.371 - Clauses incorporating cross-waivers of liability for International Space Station activities and...

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... cross-waivers of liability for International Space Station activities and Science or Space Exploration... Station activities and Science or Space Exploration activities unrelated to the International Space Station. (a) In contracts covering International Space Station activities, or Science or Space...

  15. 48 CFR 1828.371 - Clauses incorporating cross-waivers of liability for International Space Station activities and...

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... cross-waivers of liability for International Space Station activities and Science or Space Exploration... Station activities and Science or Space Exploration activities unrelated to the International Space Station. (a) In contracts covering International Space Station activities, or Science or Space...

  16. CAS-Induced Difficulties in Learning Mathematics?

    ERIC Educational Resources Information Center

    Jankvist, Uffe Thomas; Misfeldt, Morten

    2015-01-01

    In recent years computer algebra systems (CAS) have become an integrated part of the upper secondary school mathematics program. Despite the many positive possibilities of CAS, there also seems to be a flip side of the coin in relation to actual difficulties in learning mathematics, not least because a strong dependence on CAS for mathematical…

  17. CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes.

    PubMed

    Koonin, Eugene V; Makarova, Kira S

    2013-05-01

    The CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR-associated genes) is an adaptive immunity system in bacteria and archaea that functions via a distinct self-non-self recognition mechanism that is partially analogous to the mechanism of eukaryotic RNA interference (RNAi). The CRISPR-Cas system incorporates fragments of virus or plasmid DNA into the CRISPR repeat cassettes and employs the processed transcripts of these spacers as guide RNAs to cleave the cognate foreign DNA or RNA. The Cas proteins, however, are not homologous to the proteins involved in RNAi and comprise numerous, highly diverged families. The majority of the Cas proteins contain diverse variants of the RNA recognition motif (RRM), a widespread RNA-binding domain. Despite the fast evolution that is typical of the cas genes, the presence of diverse versions of the RRM in most Cas proteins provides for a simple scenario for the evolution of the three distinct types of CRISPR-cas systems. In addition to several proteins that are directly implicated in the immune response, the cas genes encode a variety of proteins that are homologous to prokaryotic toxins that typically possess nuclease activity. The predicted toxins associated with CRISPR-Cas systems include the essential Cas2 protein, proteins of COG1517 that, in addition to a ligand-binding domain and a helix-turn-helix domain, typically contain different nuclease domains and several other predicted nucleases. The tight association of the CRISPR-Cas immunity systems with predicted toxins that, upon activation, would induce dormancy or cell death suggests that adaptive immunity and dormancy/suicide response are functionally coupled. Such coupling could manifest in the persistence state being induced and potentially providing conditions for more effective action of the immune system or in cell death being triggered when immunity fails.

  18. 48 CFR 1852.228-76 - Cross-waiver of liability for international space station activities.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... for international space station activities. 1852.228-76 Section 1852.228-76 Federal Acquisition... space station activities. As prescribed in 1828.371(c) and (d), insert the following clause: Cross-waiver of liability for international space station activities (OCT 2012) (a) The...

  19. 48 CFR 1852.228-76 - Cross-waiver of liability for space station activities.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... for space station activities. 1852.228-76 Section 1852.228-76 Federal Acquisition Regulations System... CLAUSES Texts of Provisions and Clauses 1852.228-76 Cross-waiver of liability for space station activities...), insert the following clause: Cross-Waiver of Liability for Space Station Activities (DEC 1994) (a)...

  20. 48 CFR 1852.228-76 - Cross-waiver of liability for international space station activities.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... for international space station activities. 1852.228-76 Section 1852.228-76 Federal Acquisition... space station activities. As prescribed in 1828.371(c) and (d), insert the following clause: Cross-waiver of liability for international space station activities (OCT 2012) (a) The...

  1. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage

    PubMed Central

    Jiang, Fuguo; Taylor, David W.; Chen, Janice S.; Kornfeld, Jack E.; Zhou, Kaihong; Thompson, Aubri J.; Nogales, Eva; Doudna, Jennifer A.

    2016-01-01

    Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)–associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation. PMID:26841432

  2. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.

    PubMed

    Jiang, Fuguo; Taylor, David W; Chen, Janice S; Kornfeld, Jack E; Zhou, Kaihong; Thompson, Aubri J; Nogales, Eva; Doudna, Jennifer A

    2016-02-19

    Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.

  3. NASA plans and opportunities. [space flight activities throughout the 1990s

    NASA Technical Reports Server (NTRS)

    Sulzman, Frank M.

    1991-01-01

    The NASA plans for the Life Science program of a series of space flight activities throughout the decade of the 1990s are discussed with particular attention given to the NASA life science goals and objectives and to the particular space missions which will carry out these objectives. These space missions and specially designed facilities for experiments in space include Space Station Freedom, Space Biology Initiative, Gravitational Biology Facility, Life Sciences Centrifuge Facility, Controlled Ecological Life Support Systems Test Facility, and Exobiology Facility.

  4. Genome Editing in Human Cells Using CRISPR/Cas Nucleases.

    PubMed

    Wyvekens, Nicolas; Tsai, Shengdar Q; Joung, J Keith

    2015-10-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been broadly adopted for highly efficient genome editing in a variety of model organisms and human cell types. Unlike previous genome editing technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas technology does not require complex protein engineering and can be utilized by any researcher proficient in basic molecular biology and cell culture techniques. This unit describes protocols for design and cloning of vectors expressing single or multiplex gRNAs, for transient transfection of human cell lines, and for quantitation of mutation frequencies by T7 endonuclease I assay. These protocols also include guidance for using two improvements that increase the specificity of CRISPR/Cas nucleases: truncated gRNAs and dimeric RNA-guided FokI nucleases.

  5. Pyo-pneumothorax tuberculeux: à propos de 18 cas

    PubMed Central

    Hicham, Souhi; Hanane, El Ouazzani; Hicham, Janah; Ismaïl, Rhorfi; Ahmed, Abid

    2016-01-01

    Le pyo-pneumothorax tuberculeux est une complication rare mais grave de la tuberculose pulmonaire évolutive. Nous rapportons une série de 18 cas de pyo-pneumothorax tuberculeux colligés au service de Pneumo-Phtisiologie de l'Hôpital Militaire d'Instruction Mohammed V de Rabat entre janvier 2005 et décembre 2009. Il s'agit de 15 hommes et 3 femmes d’âge moyen de 35 ans ±7 ans. 4 patients étaient diabétiques. Le tabagisme était retrouvé chez 9 cas. Le pyo-pneumothorax était du coté droit dans 13 cas. La radiographie thoracique avait montré des lésions cavitaires chez 15 patients et des lésions étendues et bilatérales chez 8 cas. La recherche de BK dans le liquide de tubage gastrique était positive chez 16 cas. Un drainage thoracique associé à un traitement antituberculeux selon le régime 2SRHZ/7RH et une kinésithérapie respiratoire ont été instaurés chez tous les cas. La durée moyenne de drainage pleural était de 4 semaines. Chez 3 cas on avait noté la persistance de la suppuration pleurale ayant nécessité une toilette pleurale sous thoracoscopie avec pleurectomie et exérèse pulmonaire limitée emportant la lésion parenchymateuse tuberculeuse et la persistance d'une volumineuse poche pleurale avec trouble ventilatoire restrictif ayant nécessité une décortication pleurale chirurgicale chez deux cas. L’évolution était favorable avec pachypleurite séquellaire minime chez le reste des cas. Le pyo-pneumothorax tuberculeux est une forme grave, qui est souvent en rapport avec une tuberculose cavitaire active. L’évolution est généralement trainante malgré le traitement antituberculeux et le drainage thoracique, d'où la nécessité d'un diagnostic et un traitement précoce de toute forme de tuberculose. PMID:27583090

  6. Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos

    PubMed Central

    Tu, Zhuchi; Yang, Weili; Yan, Sen; Yin, An; Gao, Jinquan; Liu, Xudong; Zheng, Yinghui; Zheng, Jiezhao; Li, Zhujun; Yang, Su; Li, Shihua; Guo, Xiangyu; Li, Xiao-Jiang

    2017-01-01

    CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing. PMID:28155910

  7. Active disturbance rejection in large flexible space structures

    NASA Technical Reports Server (NTRS)

    Parlos, Alexander G.; Sunkel, John W.

    1990-01-01

    The design of an active control law for the rejection of persistent disturbances, in large space structures is presented. The control system design approach is based on a deterministic model of the disturbances and it optimizes the magnitude of the disturbance that the structure can tolerate without violating certain predetermined constraints. In addition to closed-loop stability, the explicit treatment of state, control, and control rate constraints, such as structural displacement and control actuator effort, guarantees that the final design will exhibit desired performance characteristics. The technique is applied to a simple two-bay truss structure, and its response is compared with that obtained using a linear-quadratic-Gaussian/loop-transfer-recovery (LQG/LTR) compensator. Preliminary results indicate that the proposed control system can reject persistent disturbances of greater magnitude by utilizing most of the available control, while limiting the structural displacements to within desired tolerances.

  8. CRISPR-Cas immunity and mobile DNA: a new superfamily of DNA transposons encoding a Cas1 endonuclease.

    PubMed

    Hickman, Alison B; Dyda, Fred

    2014-01-01

    Mobile genetic elements such as DNA transposons are a feature of most genomes. The existence of novel DNA transposons can be inferred when whole genome sequencing reveals the presence of hallmarks of mobile elements such as terminal inverted repeats (TIRs) flanked by target site duplications (TSDs). A recent report describes a new superfamily of DNA transposons in the genomes of a few bacteria and archaea that possess TIRs and TSDs, and encode several conserved genes including a cas1 endonuclease gene, previously associated only with CRISPR-Cas adaptive immune systems. The data strongly suggests that these elements, designated 'casposons', are likely to be bona fide DNA transposons and that their Cas1 nucleases act as transposases and are possibly still active.

  9. CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo

    PubMed Central

    Moreno-Mateos, Miguel A.; Vejnar, Charles E.; Beaudoin, Jean-Denis; Fernandez, Juan P.; Mis, Emily K.; Khokha, Mustafa K.; Giraldez, Antonio J.

    2015-01-01

    CRISPR/Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We have analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observe that guanine enrichment and adenine depletion increase sgRNA stability and activity, while loading, nucleosome positioning and Cas9 off-target binding are not major determinants. We additionally identified truncated and 5′ mismatch-containing sgRNAs as efficient alternatives to canonical sgRNAs. Based on these results, we created a predictive sgRNA-scoring algorithm (CRISPRscan.org) that effectively captures the sequence features affecting Cas9/sgRNA activity in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos-3′-UTR fusion can generate maternal-zygotic mutants, increase viability and reduce somatic mutations. Together, these results provide novel insights into the determinants that influence Cas9 activity and a framework to identify highly efficient sgRNAs for genome targeting in vivo. PMID:26322839

  10. Looking at Earth from Space: Teacher's Guide with Activities for Earth and Space Science

    NASA Technical Reports Server (NTRS)

    Steele, Colleen (Editor); Steele, Colleen; Ryan, William F.

    1995-01-01

    The Maryland Pilot Earth Science and Technology Education Network (MAPS-NET) project was sponsored by the National Aeronautics and Space Administration (NASA) to enrich teacher preparation and classroom learning in the area of Earth system science. This publication includes a teacher's guide that replicates material taught during a graduate-level course of the project and activities developed by the teachers. The publication was developed to provide teachers with a comprehensive approach to using satellite imagery to enhance science education. The teacher's guide is divided into topical chapters and enables teachers to expand their knowledge of the atmosphere, common weather patterns, and remote sensing. Topics include: weather systems and satellite imagery including mid-latitude weather systems; wave motion and the general circulation; cyclonic disturbances and baroclinic instability; clouds; additional common weather patterns; satellite images and the internet; environmental satellites; orbits; and ground station set-up. Activities are listed by suggested grade level and include the following topics: using weather symbols; forecasting the weather; cloud families and identification; classification of cloud types through infrared Automatic Picture Transmission (APT) imagery; comparison of visible and infrared imagery; cold fronts; to ski or not to ski (imagery as a decision making tool), infrared and visible satellite images; thunderstorms; looping satellite images; hurricanes; intertropical convergence zone; and using weather satellite images to enhance a study of the Chesapeake Bay. A list of resources is also included.

  11. Light Echoes and Cold Dust in Cas A

    NASA Astrophysics Data System (ADS)

    Krause, O.; Rieke, G. H.; Birkmann, S.

    2006-06-01

    We report on infrared observations of the prototypical supernova remnant Cassiopeia A obtained with the Spitzer Space Telescope. Two images of Cas A taken at 24 micrometers with the MIPS instrument over a 1-year time interval revealed moving structures outside the shell of the supernova remnant to a distance of more than 20 arc minutes. The observed tangential velocities are at roughly the speed of light. The moving structures are infrared echoes, in which interstellar dust is heated by the explosion and by flares from the compact object near the center of the remnant. Far-infrared maps of the remnant at 160 micrometers in combination with molecular line observations demonstrate that most of recently detected submillimetre emission towards Cas A originates from interstellar dust in a molecular cloud complex located in the line of sight between the Earth and the remnant, rather than from a large amount (about three solar masses) of cold (18K) dust within Cas A. The argument that type II supernovae produce copious amounts of dust is therefore not supported by the case of Cas A, which previously appeared to provide the best evidence for this possibility.

  12. Active Volcano Monitoring using a Space-based Hyperspectral Imager

    NASA Astrophysics Data System (ADS)

    Cipar, J. J.; Dunn, R.; Cooley, T.

    2010-12-01

    Active volcanoes occur on every continent, often in close proximity to heavily populated areas. While ground-based studies are essential for scientific research and disaster mitigation, remote sensing from space can provide rapid and continuous monitoring of active and potentially active volcanoes [Ramsey and Flynn, 2004]. In this paper, we report on hyperspectral measurements of Kilauea volcano, Hawaii. Hyperspectral images obtained by the US Air Force TacSat-3/ARTEMIS sensor [Lockwood et al, 2006] are used to obtain estimates of the surface temperatures for the volcano. ARTEMIS measures surface-reflected light in the visible, near-infrared, and short-wave infrared bands (VNIR-SWIR). The SWIR bands are known to be sensitive to thermal radiation [Green, 1996]. For example, images from the NASA Hyperion hyperspectral sensor have shown the extent of wildfires and active volcanoes [Young, 2009]. We employ the methodology described by Dennison et al, (2006) to obtain an estimate of the temperature of the active region of Kilauea. Both day and night-time images were used in the analysis. To improve the estimate, we aggregated neighboring pixels. The active rim of the lava lake is clearly discernable in the temperature image, with a measured temperature exceeding 1100o C. The temperature decreases markedly on the exterior of the summit crater. While a long-wave infrared (LWIR) sensor would be ideal for volcano monitoring, we have shown that the thermal state of an active volcano can be monitored using the SWIR channels of a reflective hyperspectral imager. References: Dennison, Philip E., Kraivut Charoensiri, Dar A. Roberts, Seth H. Peterson, and Robert O. Green (2006). Wildfire temperature and land cover modeling using hyperspectral data, Remote Sens. Environ., vol. 100, pp. 212-222. Green, R. O. (1996). Estimation of biomass fire temperature and areal extent from calibrated AVIRIS spectra, in Summaries of the 6th Annual JPL Airborne Earth Science Workshop, Pasadena, CA

  13. Collision Avoidance System (CAS): Human Factors Engineering Evaluation.

    DTIC Science & Technology

    1982-12-01

    personnel indicated that the CAS console was much too big for the limited amount of space available on RANGER’s bridge. The console is 40 inches wide...avoidance (C/A) data. > RANGE * 12//2 < (T >~ LOG SPEED 4 10.0 KT < E1 i >~ HEADING 4 270 DEG * < J [ > BRGCRSR * 000 DEG4C/ ADATA < - > TRIAL SPD 4 0 <EJ

  14. Time-dependent restricted-active-space self-consistent-field theory with space partition

    NASA Astrophysics Data System (ADS)

    Miyagi, Haruhide; Madsen, Lars Bojer

    2017-02-01

    Aiming at efficient numerical analysis of time-dependent (TD) many-electron dynamics of atoms involving multielectron continua, the TD restricted-active-space self-consistent-field theory with space partition (TD-RASSCF-SP) is presented. The TD-RASSCF-SP wave function is expanded in terms of TD configuration-interaction coefficients with Slater determinants composed of two kinds of TD orbitals: M ̂ orbitals are defined to be nonvanishing in the inner region (V ̂), a small volume around the atomic nucleus, and M ˇ orbitals are nonvanishing in the large outer region (V ˇ). For detailed discussion of the SP strategy, the equations of motion are derived by two different formalisms for comparison. To ensure continuous differentiability of the wave function across the two regions, one of the formalisms makes use of the property of the finite-element discrete-variable-representation (FEDVR) functions and introduces additional time-independent orbitals. The other formalism is more general and is based on the Bloch operator as in the R -matrix theory, but turns out to be less practical for numerical applications. Hence, using the FEDVR-based formalism, the numerical performance is tested by computing double-ionization dynamics of atomic beryllium in intense light fields. To achieve high accuracy, M ̂ should be set large to take into account the strong many-electron correlation around the nucleus. On the other hand, M ˇ can be set much smaller than M ̂ for capturing the weaker correlation between the two outgoing photoelectrons. As a result, compared with more accurate multiconfigurational TD Hartree-Fock (MCTDHF) method, the TD-RASSCF-SP method may achieve comparable accuracy in the description of the double-ionization dynamics. There are, however, difficulties related to the stiffness of the equations of motion of the TD-RASSCF-SP method, which makes the required time step for this method smaller than the one needed for the MCTDHF approach.

  15. STRUCTURAL BIOLOGY. A Cas9-guide RNA complex preorganized for target DNA recognition.

    PubMed

    Jiang, Fuguo; Zhou, Kaihong; Ma, Linlin; Gressel, Saskia; Doudna, Jennifer A

    2015-06-26

    Bacterial adaptive immunity uses CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR transcripts for foreign DNA degradation. In type II CRISPR-Cas systems, activation of Cas9 endonuclease for DNA recognition upon guide RNA binding occurs by an unknown mechanism. Crystal structures of Cas9 bound to single-guide RNA reveal a conformation distinct from both the apo and DNA-bound states, in which the 10-nucleotide RNA "seed" sequence required for initial DNA interrogation is preordered in an A-form conformation. This segment of the guide RNA is essential for Cas9 to form a DNA recognition-competent structure that is poised to engage double-stranded DNA target sequences. We construe this as convergent evolution of a "seed" mechanism reminiscent of that used by Argonaute proteins during RNA interference in eukaryotes.

  16. Spatial Polygamy and Contextual Exposures (SPACEs): Promoting Activity Space Approaches in Research on Place and Health

    PubMed Central

    Matthews, Stephen A.; Yang, Tse-Chuan

    2014-01-01

    Exposure science has developed rapidly and there is an increasing call for greater precision in the measurement of individual exposures across space and time. Social science interest in an individual’s environmental exposure, broadly conceived, has arguably been quite limited conceptually and methodologically. Indeed, we appear to lag behind our exposure science colleagues in our theories, data, and methods. In this paper we discuss a framework based on the concept of spatial polygamy to demonstrate the need to collect new forms of data on human spatial behavior and contextual exposures across time and space. Adopting new data and methods will be essential if we want to better understand social inequality in terms of exposure to health risks and access to health resources. We discuss the opportunities and challenges focusing on the potential seemingly offered by focusing on human mobility, and specifically the utilization of activity space concepts and data. A goal of the paper is to spatialize social and health science concepts and research practice vis-a-vis the complexity of exposure. The paper concludes with some recommendations for future research focusing on theoretical and conceptual development, promoting research on new types of places and human movement, the dynamic nature of contexts, and on training. “When we elect wittingly or unwittingly, to work within a level … we tend to discern or construct – whichever emphasis you prefer – only those kinds of systems whose elements are confined to that level.”Otis Dudley Duncan (1961, p. 141). “…despite the new ranges created by improved transportation, local government units have tended to remain medieval in size.”Torsten Hägerstrand (1970, p.18) “A detective investigating a crime needs both tools and understanding. If he has no fingerprint powder, he will fail to find fingerprints on most surfaces. If he does not understand where the criminal is likely to have put his fingers, he will not

  17. Spatial Polygamy and Contextual Exposures (SPACEs): Promoting Activity Space Approaches in Research on Place and Health.

    PubMed

    Matthews, Stephen A; Yang, Tse-Chuan

    2013-08-01

    Exposure science has developed rapidly and there is an increasing call for greater precision in the measurement of individual exposures across space and time. Social science interest in an individual's environmental exposure, broadly conceived, has arguably been quite limited conceptually and methodologically. Indeed, we appear to lag behind our exposure science colleagues in our theories, data, and methods. In this paper we discuss a framework based on the concept of spatial polygamy to demonstrate the need to collect new forms of data on human spatial behavior and contextual exposures across time and space. Adopting new data and methods will be essential if we want to better understand social inequality in terms of exposure to health risks and access to health resources. We discuss the opportunities and challenges focusing on the potential seemingly offered by focusing on human mobility, and specifically the utilization of activity space concepts and data. A goal of the paper is to spatialize social and health science concepts and research practice vis-a-vis the complexity of exposure. The paper concludes with some recommendations for future research focusing on theoretical and conceptual development, promoting research on new types of places and human movement, the dynamic nature of contexts, and on training. "When we elect wittingly or unwittingly, to work within a level … we tend to discern or construct - whichever emphasis you prefer - only those kinds of systems whose elements are confined to that level."Otis Dudley Duncan (1961, p. 141)."…despite the new ranges created by improved transportation, local government units have tended to remain medieval in size."Torsten Hägerstrand (1970, p.18)"A detective investigating a crime needs both tools and understanding. If he has no fingerprint powder, he will fail to find fingerprints on most surfaces. If he does not understand where the criminal is likely to have put his fingers, he will not look in the right

  18. Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization

    PubMed Central

    Senturk, Serif; Shirole, Nitin H.; Nowak, Dawid G.; Corbo, Vincenzo; Pal, Debjani; Vaughan, Alexander; Tuveson, David A.; Trotman, Lloyd C.; Kinney, Justin B.; Sordella, Raffaella

    2017-01-01

    The CRISPR/Cas9 system is a powerful tool for studying gene function. Here, we describe a method that allows temporal control of CRISPR/Cas9 activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9) enables conditional Cas9 expression and temporal control of gene editing in the presence of an FKBP12 synthetic ligand. This system can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest without co-modulation of the latter. In particular, when co-expressed with inducible Cre-ERT2, our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with single-cell specificity. We anticipate this platform will be used for the systematic characterization and identification of essential genes, as well as the investigation of the interactions between functional genes. PMID:28224990

  19. Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System.

    PubMed

    Redding, Sy; Sternberg, Samuel H; Marshall, Myles; Gibb, Bryan; Bhat, Prashant; Guegler, Chantal K; Wiedenheft, Blake; Doudna, Jennifer A; Greene, Eric C

    2015-11-05

    CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease helicase for target degradation. Here, we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways dictated by the presence or absence of a protospacer-adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short single-stranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent on the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes and support a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.

  20. Surveillance and processing of foreign DNA by the Escherichia coli CRISPR-Cas system

    PubMed Central

    Redding, Sy; Sternberg, Samuel H.; Marshall, Myles; Gibb, Bryan; Bhat, Prashant; Guegler, Chantal K.; Wiedenheft, Blake; Doudna, Jennifer A.; Greene, Eric C.

    2015-01-01

    Summary CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease-helicase for target degradation. Here we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways, dictated by the presence or absence of a protospacer adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short singlestranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent upon the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes, and supports a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA. PMID:26522594

  1. Utilisation of Wearable Computing for Space Programmes Test Activities Optimasation

    NASA Astrophysics Data System (ADS)

    Basso, V.; Lazzari, D.; Alemanni, M.

    2004-08-01

    New technologies are assuming a relevant importance in the Space business domain also in the Assembly Integration and Test (AIT) activities allowing process optimization and capability that were unthinkable only few years ago. This paper has the aim to describe Alenia Spazio (ALS) gained experience on the remote interaction techniques as a results of collaborations established both on European Communities (EC) initiatives, with Alenia Aeronautica (ALA) and Politecnico of Torino (POLITO). The H/W and S/W components performances increase and costs reduction due to the home computing massive utilization (especially demanded by the games business) together with the network technology possibility (offered by the web as well as the hi-speed links and the wireless communications) allow today to re-think the traditional AIT process activities in the light of the multimedia data exchange: graphical, voice video and by sure more in the future. Aerospace business confirm its innovation vocation which in the year '80 represents the cradle of the CAD systems and today is oriented to the 3D data visualization/ interaction technologies and remote visualisation/ interaction in collaborative way on a much more user friendly bases (i.e. not for specialists). Fig. 1 collects AIT extended scenario studied and adopted by ALS in these years. ALS experimented two possibilities of remote visualization/interaction: Portable [e.g. Fig.2 Personal Digital Assistant (PDA), Wearable] and walls (e.g.VR-Lab) screens as both 2D/3D visualisation and interaction devices which could support many types of traditional (mainly based on EGSE and PDM/CAD utilisation/reports) company internal AIT applications: 1. design review support 2. facility management 3. storage management 4. personnel training 5. integration sequences definition 6. assembly and test operations follow up 7. documentation review and external access to AIT activities for remote operations (e.g. tele-testing) EGSE Portable Clean room

  2. CRISPR/Cas9 Technologies.

    PubMed

    Williams, Bart O; Warman, Matthew L

    2017-02-23

    The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) pathway is revolutionizing biological research. Modifications to this primitive prokaryotic immune system now enable scientists to efficiently edit DNA or modulate gene expression in living eukaryotic cells and organisms. Thus, many laboratories can now perform important experiments that previously were considered scientifically risky or too costly. Here, we describe the components of the CRISPR/Cas system that have been engineered for use in eukaryotes. We also explain how this system can be used to genetically modify cell lines and model organisms, or regulate gene expression in order to search for new participants in biological pathways. © 2017 American Society for Bone and Mineral Research.

  3. The Aspergillus fumigatus metacaspases CasA and CasB facilitate growth under conditions of endoplasmic reticulum stress.

    PubMed

    Richie, Daryl L; Miley, Michael D; Bhabhra, Ruchi; Robson, Geoffrey D; Rhodes, Judith C; Askew, David S

    2007-01-01

    We have examined the contribution of metacaspases to the growth and stress response of the opportunistic human mould pathogen, Aspergillus fumigatus, based on increasing evidence implicating the yeast metacaspase Yca1p in apoptotic-like programmed cell death. Single metacaspase-deficient mutants were constructed by targeted disruption of each of the two metacaspase genes in A. fumigatus, casA and casB, and a metacaspase-deficient mutant, DeltacasA/DeltacasB, was constructed by disrupting both genes. Stationary phase cultures of wild-type A. fumigatus were associated with the appearance of typical markers of apoptosis, including elevated proteolytic activity against caspase substrates, phosphatidylserine exposure on the outer leaflet of the membrane, and loss of viability. By contrast, phosphatidylserine exposure was not observed in stationary phase cultures of the DeltacasA/DeltacasB mutant, although caspase activity and viability was indistinguishable from wild type. The mutant retained wild-type virulence and showed no difference in sensitivity to a range of pro-apoptotic stimuli that have been reported to initiate yeast apoptosis. However, the DeltacasA/DeltacasB mutant showed a growth detriment in the presence of agents that disrupt endoplasmic reticulum homeostasis. These findings demonstrate that metacaspase activity in A. fumigatus contributes to the apoptotic-like loss of membrane phospholipid asymmetry at stationary phase, and suggest that CasA and CasB have functions that support growth under conditions of endoplasmic reticulum stress.

  4. Long-Term International Space Station (ISS) Risk Reduction Activities

    NASA Technical Reports Server (NTRS)

    Forroci, Michael P.; Gafka, George K.; Lutomski, Michael G.; Maher, Jacilyn S.

    2011-01-01

    As the assembly of the ISS nears completion, it is worthwhile to step back and review some of the actions pursued by the Program in recent years to reduce risk and enhance the safety and health of ISS crewmembers, visitors, and space flight participants. While the initial ISS requirements and design were intended to provide the best practicable levels of safety, it is always possible to further reduce risk given the determination, commitment, and resources to do so. The following is a summary of some of the steps taken by the ISS Program Manager, by our International Partners, by hardware and software designers, by operational specialists, and by safety personnel to continuously enhance the safety of the ISS, and to reduce risk to all crewmembers. While years of work went into the development of ISS requirements, there are many things associated with risk reduction in a Program like the ISS that can only be learned through actual operational experience. These risk reduction activities can be divided into roughly three categories: Areas that were initially noncompliant which have subsequently been brought into compliance or near compliance (i.e., Micrometeoroid and Orbital Debris [MMOD] protection, acoustics) Areas where initial design requirements were eventually considered inadequate and were subsequently augmented (i.e., Toxicity hazard level-4 materials, emergency procedures, emergency equipment, control of drag-throughs) Areas where risks were initially underestimated, and have subsequently been addressed through additional mitigation (i.e., Extravehicular Activity [EVA] sharp edges, plasma shock hazards). Due to the hard work and cooperation of many parties working together across the span of more than a decade, the ISS is now a safer and healthier environment for our crew, in many cases exceeding the risk reduction targets inherent in the intent of the original design. It will provide a safe and stable platform for utilization and discovery for years to come.

  5. [Activities of Space Telescope Science Institute with the Hubble Space Telescope

    NASA Technical Reports Server (NTRS)

    Dempsey, Robert C.; Neff, James E.; Strassmeier, Klaus G.; Linsky, Jeffrey L.

    1998-01-01

    A number of studies, especially in recent years with the Hubble Space Telescope's (HST) Goddard High Resolution Spectrograph (GHRS), have been presented on the UV line profiles of late-type stars. Generally, these consist of a few "snapshot" spectra of several different key diagnostic emission lines. From this it has become clear that many active stars possess non-gaussian line profiles. Unlike the case with AR Lac, observed with IUE, no assymetric profile has been clearly identified that results from an inhomogeneous surface temperature or density distribution. In 1993 we attempted to observe the RS CVn binary V711 Tau at several phases with the GHRS in a number of UV bandpasses in order to study profile variations as a function of phase. Unfortunately, scheduling problems, pointing errors, continuous flaring and the sparse and uneven phase sampling prevented us from achieving the primary goal. However, it is clear that a number of UV lines in the system, notably C IV, Si IV and Mg II show very extended emission out to several hundred km/s. The profiles were also clearly variable. Vilhu et al. (1997) and Walter et al. (1995) conducted a campaign on the rapidly rotating, single star AB Dor, where they observed C IV continuously for 14 hours. They found extended, non-gaussian emission in the C IV doublet and that Doppler images derived from these images were remarkably similar to the simultaneous spot-image. In a follow up study of V711 Tau we have observed another RS CVn with complete phase coverage in three key wavelength bandpasses, utilizing the ability of HST to observe some stars at high latitudes in uninterrupted fashion. Generally classified as an RS CVn, V824 Ara (HD 155555) consists of a G5 IV star in a short period orbit (P=ld.68) with a K0 V-IV companion. However, the system does not eclipse and therefore does not rigorously fit the Hall (1976) definition. There is also a visual M star companion (LDS587B) 33 arcsec away. The space velocities of the stars

  6. Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9

    PubMed Central

    Feng, Yan; Chen, Cheng; Han, Yuxiang; Chen, Zelin; Lu, Xiaochan; Liang, Fang; Li, Song; Qin, Wei; Lin, Shuo

    2016-01-01

    The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5′-NGG-3′ protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology. PMID:27317783

  7. The association between objectively measured physical activity and life-space mobility among older people.

    PubMed

    Tsai, L-T; Portegijs, E; Rantakokko, M; Viljanen, A; Saajanaho, M; Eronen, J; Rantanen, T

    2015-08-01

    The purpose of this cross-sectional study was to investigate the association between objectively measured physical activity and life-space mobility in community-dwelling older people. Life-space refers to the spatial area a person purposefully moves through in daily life (bedroom, home, yard, neighborhood, town, and beyond) and life-space mobility to the frequency of travel and the help needed when moving through different life-space areas. The study population comprised community-living 75- to 90-year-old people {n = 174; median age 79.7 [interquartile range (IQR) 7.1]}, participating in the accelerometer substudy of Life-Space Mobility in Old Age (LISPE) project. Step counts and activity time were measured by an accelerometer (Hookie "AM20 Activity Meter") for 7 days. Life-space mobility was assessed with Life-Space Assessment (LSA) questionnaire. Altogether, 16% had a life-space area restricted to the neighborhood when moving independently. Participants with a restricted life space were less physically active and about 70% of them had exceptionally low values in daily step counts (≤ 615 steps) and moderate activity time (≤ 6.8 min). Higher step counts and activity time correlated positively with life-space mobility. Prospective studies are needed to clarify the temporal order of low physical activity level and restriction in life-space mobility.

  8. 48 CFR 1852.228-78 - Cross-waiver of liability for science or space exploration activities unrelated to the...

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... for science or space exploration activities unrelated to the International Space Station. 1852.228-78... Cross-waiver of liability for science or space exploration activities unrelated to the International... Liability for Science or Space Exploration Activities Unrelated to the International Space Station (OCT...

  9. 48 CFR 1852.228-78 - Cross-waiver of liability for science or space exploration activities unrelated to the...

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... for science or space exploration activities unrelated to the International Space Station. 1852.228-78... Cross-waiver of liability for science or space exploration activities unrelated to the International... Liability for Science or Space Exploration Activities Unrelated to the International Space Station (OCT...

  10. Public open space, physical activity, urban design and public health: Concepts, methods and research agenda.

    PubMed

    Koohsari, Mohammad Javad; Mavoa, Suzanne; Villanueva, Karen; Sugiyama, Takemi; Badland, Hannah; Kaczynski, Andrew T; Owen, Neville; Giles-Corti, Billie

    2015-05-01

    Public open spaces such as parks and green spaces are key built environment elements within neighbourhoods for encouraging a variety of physical activity behaviours. Over the past decade, there has been a burgeoning number of active living research studies examining the influence of public open space on physical activity. However, the evidence shows mixed associations between different aspects of public open space (e.g., proximity, size, quality) and physical activity. These inconsistencies hinder the development of specific evidence-based guidelines for urban designers and policy-makers for (re)designing public open space to encourage physical activity. This paper aims to move this research agenda forward, by identifying key conceptual and methodological issues that may contribute to inconsistencies in research examining relations between public open space and physical activity.

  11. Aeronautics and Space Report of the President: 1977 Activities.

    ERIC Educational Resources Information Center

    National Aeronautics and Space Administration, Washington, DC.

    The national programs in aeronautics and space made steady progress in 1977 toward their long-term objectives. In aeronautics the goals were improved performance, energy efficiency, and safety in aircraft. In space the goals were: (1) better remote sensing systems to generate more sophisticated information about the Earth's environment; (2)…

  12. Medical operations and life sciences activities on space station

    NASA Technical Reports Server (NTRS)

    Johnson, P. C. (Editor); Mason, J. A. (Editor)

    1982-01-01

    Space station health maintenance facilities, habitability, personnel, and research in the medical sciences and in biology are discussed. It is assumed that the space station structure will consist of several modules, each being consistent with Orbiter payload bay limits in size, weight, and center of gravity.

  13. Planetary Data Archiving Activities in Indian Space Research Organisation (isro)

    NASA Astrophysics Data System (ADS)

    Gopala Krishna, Barla; Srivastava, Pradeep Kumar

    The Indian Space Research Organisation (ISRO) has launched its first planetary mission to Moon viz., Chandrayaan-1 on October 22, 2008. The basic objectives of the Chandrayaan-1 mission are photoselenological and chemical mapping of the Moon with improved spatial and spectral resolution. The payloads in this mission are: (i) Terrain mapping stereo camera (TMC) with 20km swath (400-900 nm band) for 3D imaging of lunar surface at a spatial resolution of 5m (ii) Hyper Spectral Imager (HySI) in the 400-920 nm band with 64 channels and spatial resolution of 80m (20km swath) for mineralogical mapping (iii) High-energy X-ray (30-270 keV) spectrometer having a footprint of 40km for study of volatile transport on Moon and (iv) Laser ranging instrument with vertical resolution of 5m (v) Miniature imaging radar instrument (Mini-SAR) from APL, NASA to look for presence of ice in the polar region (vi) Near infrared spectrometer (SIR-2) from Max Plank Institute, Germany (vii)Moon Mineralogy Mapper (M3) from JPL, NASA for mineralogical mapping in the infra-red regions (0.7 -3.0 micron) (viii) Sub-keV Atom Reflecting Analyzer (SARA) from Sweden, India and Japan for detection of low energy neutral atoms emanated from the lunar surface (ix) Radiation Dose Monitor (RADOM) from Bulgaria for monitoring energetic particle flux in the lunar environment and (x) Collimated low energy (1-10keV) X-ray spectrometer (C1XS) with a field of view of 20km for chemical mapping of the lunar surface from RAL, UK. A wealth of data has been collected (November 2008 to August 2009) from the above instru-ments during the mission life of Chandrayaan-1 and the science data from these instruments is being archived at Indian Space Science Data Centre (ISSDC). ISRO Science Data Archive (ISDA) identified at ISSDC is the primary data archive for the payload data of current and future Indian space science missions. The data center (ISSDC) is responsible for the Ingest, Archive, and Dissemination of the payload

  14. Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome

    PubMed Central

    Müller, Maximilian; Lee, Ciaran M; Gasiunas, Giedrius; Davis, Timothy H; Cradick, Thomas J; Siksnys, Virginijus; Bao, Gang; Cathomen, Toni; Mussolino, Claudio

    2016-01-01

    RNA-guided nucleases (RGNs) based on the type II CRISPR-Cas9 system of Streptococcus pyogenes (Sp) have been widely used for genome editing in experimental models. However, the nontrivial level of off-target activity reported in several human cells may hamper clinical translation. RGN specificity depends on both the guide RNA (gRNA) and the protospacer adjacent motif (PAM) recognized by the Cas9 protein. We hypothesized that more stringent PAM requirements reduce the occurrence of off-target mutagenesis. To test this postulation, we generated RGNs based on two Streptococcus thermophilus (St) Cas9 proteins, which recognize longer PAMs, and performed a side-by-side comparison of the three RGN systems targeted to matching sites in two endogenous human loci, PRKDC and CARD11. Our results demonstrate that in samples with comparable on-target cleavage activities, significantly lower off-target mutagenesis was detected using St-based RGNs as compared to the standard Sp-RGNs. Moreover, similarly to SpCas9, the StCas9 proteins accepted truncated gRNAs, suggesting that the specificities of St-based RGNs can be further improved. In conclusion, our results show that Cas9 proteins with longer or more restrictive PAM requirements provide a safe alternative to SpCas9-based RGNs and hence a valuable option for future human gene therapy applications. PMID:26658966

  15. Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch

    PubMed Central

    Oakes, Benjamin L; Nadler, Dana C.; Flamholz, Avi; Fellmann, Christof; Staahl, Brett T.; Doudna, Jennifer A.; Savage, David F.

    2016-01-01

    The CRISPR-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disrupting the enzyme’s binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the Estrogen Receptor α Ligand Binding Domain. This protein displayed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9. PMID:27136077

  16. Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition

    PubMed Central

    Rollie, Clare; Schneider, Stefanie; Brinkmann, Anna Sophie; Bolt, Edward L; White, Malcolm F

    2015-01-01

    The adaptive prokaryotic immune system CRISPR-Cas provides RNA-mediated protection from invading genetic elements. The fundamental basis of the system is the ability to capture small pieces of foreign DNA for incorporation into the genome at the CRISPR locus, a process known as Adaptation, which is dependent on the Cas1 and Cas2 proteins. We demonstrate that Cas1 catalyses an efficient trans-esterification reaction on branched DNA substrates, which represents the reverse- or disintegration reaction. Cas1 from both Escherichia coli and Sulfolobus solfataricus display sequence specific activity, with a clear preference for the nucleotides flanking the integration site at the leader-repeat 1 boundary of the CRISPR locus. Cas2 is not required for this activity and does not influence the specificity. This suggests that the inherent sequence specificity of Cas1 is a major determinant of the adaptation process. DOI: http://dx.doi.org/10.7554/eLife.08716.001 PMID:26284603

  17. Climate Change Adaptation Science Activities at NASA Johnson Space Center

    NASA Technical Reports Server (NTRS)

    Stefanov, William L.; Lulla, Kamlesh

    2012-01-01

    The Johnson Space Center (JSC), located in the southeast metropolitan region of Houston, TX is the prime NASA center for human spaceflight operations and astronaut training, but it also houses the unique collection of returned extraterrestrial samples, including lunar samples from the Apollo missions. The Center's location adjacent to Clear Lake and the Clear Creek watershed, an estuary of Galveston Bay, puts it at direct annual risk from hurricanes, but also from a number of other climate-related hazards including drought, floods, sea level rise, heat waves, and high wind events all assigned Threat Levels of 2 or 3 in the most recent NASA Center Disaster/Risk Matrix produced by the Climate Adaptation Science Investigator Working Group. Based on prior CASI workshops at other NASA centers, it is recognized that JSC is highly vulnerable to climate-change related hazards and has a need for adaptation strategies. We will present an overview of prior CASI-related work at JSC, including publication of a climate change and adaptation informational data brochure, and a Resilience and Adaptation to Climate Risks Workshop that was held at JSC in early March 2012. Major outcomes of that workshop that form a basis for work going forward are 1) a realization that JSC is embedded in a regional environmental and social context, and that potential climate change effects and adaptation strategies will not, and should not, be constrained by the Center fence line; 2) a desire to coordinate data collection and adaptation planning activities with interested stakeholders to form a regional climate change adaptation center that could facilitate interaction with CASI; 3) recognition that there is a wide array of basic data (remotely sensed, in situ, GIS/mapping, and historical) available through JSC and other stakeholders, but this data is not yet centrally accessible for planning purposes.

  18. What rheumatologists need to know about CRISPR/Cas9.

    PubMed

    Gibson, Gary J; Yang, Maozhou

    2017-02-09

    CRISPR/Cas9 genome editing technology has taken the research world by storm since its use in eukaryotes was first proposed in 2012. Publications describing advances in technology and new applications have continued at an unrelenting pace since that time. In this Review, we discuss the application of CRISPR/Cas9 for creating gene mutations - the application that initiated the current avalanche of interest - and new developments that have largely answered initial concerns about its specificity and ability to introduce new gene sequences. We discuss the new, diverse and rapidly growing adaptations of the CRISPR/Cas9 technique that enable activation, repression, multiplexing and gene screening. These developments have enabled researchers to create sophisticated tools for dissecting the function and inter-relatedness of genes, as well as noncoding regions of the genome, and to identify gene networks and noncoding regions that promote disease or confer disease susceptibility. These approaches are beginning to be used to interrogate complex and multilayered biological systems and to produce complex animal models of disease. CRISPR/Cas9 technology has enabled the application of new therapeutic approaches to treating disease in animal models, some of which are beginning to be seen in the first human clinical trials. We discuss the direct application of these techniques to rheumatic diseases, which are currently limited but are sure to increase rapidly in the near future.

  19. CRISPR-Cas9: from Genome Editing to Cancer Research

    PubMed Central

    Chen, Si; Sun, Heng; Miao, Kai; Deng, Chu-Xia

    2016-01-01

    Cancer development is a multistep process triggered by innate and acquired mutations, which cause the functional abnormality and determine the initiation and progression of tumorigenesis. Gene editing is a widely used engineering tool for generating mutations that enhance tumorigenesis. The recent developed clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9) system renews the genome editing approach into a more convenient and efficient way. By rapidly introducing genetic modifications in cell lines, organs and animals, CRISPR-Cas9 system extends the gene editing into whole genome screening, both in loss-of-function and gain-of-function manners. Meanwhile, the system accelerates the establishment of animal cancer models, promoting in vivo studies for cancer research. Furthermore, CRISPR-Cas9 system is modified into diverse innovative tools for observing the dynamic bioprocesses in cancer studies, such as image tracing for targeted DNA, regulation of transcription activation or repression. Here, we view recent technical advances in the application of CRISPR-Cas9 system in cancer genetics, large-scale cancer driver gene hunting, animal cancer modeling and functional studies. PMID:27994508

  20. CRISPR-Cas9: from Genome Editing to Cancer Research.

    PubMed

    Chen, Si; Sun, Heng; Miao, Kai; Deng, Chu-Xia

    2016-01-01

    Cancer development is a multistep process triggered by innate and acquired mutations, which cause the functional abnormality and determine the initiation and progression of tumorigenesis. Gene editing is a widely used engineering tool for generating mutations that enhance tumorigenesis. The recent developed clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9) system renews the genome editing approach into a more convenient and efficient way. By rapidly introducing genetic modifications in cell lines, organs and animals, CRISPR-Cas9 system extends the gene editing into whole genome screening, both in loss-of-function and gain-of-function manners. Meanwhile, the system accelerates the establishment of animal cancer models, promoting in vivo studies for cancer research. Furthermore, CRISPR-Cas9 system is modified into diverse innovative tools for observing the dynamic bioprocesses in cancer studies, such as image tracing for targeted DNA, regulation of transcription activation or repression. Here, we view recent technical advances in the application of CRISPR-Cas9 system in cancer genetics, large-scale cancer driver gene hunting, animal cancer modeling and functional studies.

  1. Destination state screening of active spaces in spin dynamics simulations

    NASA Astrophysics Data System (ADS)

    Krzystyniak, M.; Edwards, Luke J.; Kuprov, Ilya

    2011-06-01

    We propose a novel avenue for state space reduction in time domain Liouville space spin dynamics simulations, using detectability as a selection criterion - only those states that evolve into or affect other detectable states are kept in the simulation. This basis reduction procedure (referred to as destination state screening) is formally exact and can be applied on top of the existing state space restriction techniques. As demonstrated below, in many cases this results in further reduction of matrix dimension, leading to considerable acceleration of many spin dynamics simulation types. Destination state screening is implemented in the latest version of the Spinach library (http://spindynamics.org).

  2. Campylobacter jejuni acquire new host-derived CRISPR spacers when in association with bacteriophages harboring a CRISPR-like Cas4 protein

    PubMed Central

    Hooton, Steven P. T.; Connerton, Ian F.

    2015-01-01

    Campylobacter jejuni is a worldwide cause of human diarrhoeal disease. Clustered Repetitively Interspaced Palindromic Repeats (CRISPRs) and associated proteins allow Bacteria and Archaea to evade bacteriophage and plasmid infection. Type II CRISPR systems are found in association with combinations of genes encoding the CRISPR-associated Cas1, Cas2, Cas4 or Csn2, and Cas9 proteins. C. jejuni possesses a minimal subtype II-C CRISPR system containing cas1, cas2, and cas9 genes whilst cas4 is notably absent. Cas4 proteins possess 5′-3′ exonuclease activity to create recombinogenic-ends for spacer acquisition. Here we report a conserved Cas4-like protein in Campylobacter bacteriophages that creates a novel split arrangement between the bacteriophage and host that represents a new twist in the bacteriophage/host co-evolutionary arms race. The continuous association of bacteriophage and host in the carrier state life cycle of C. jejuni provided an opportunity to study spacer acquisition in this species. Remarkably all the spacer sequences observed were of host origin. We hypothesize that Campylobacter bacteriophages can use Cas4-like protein to activate spacer acquisition to use host DNA as an effective decoy to bacteriophage DNA. Bacteria that acquire self-spacers and escape phage infection must overcome CRISPR-mediated autoimmunity either by loss of the interference functions leaving them susceptible to foreign DNA incursion or tolerate changes in gene regulation. PMID:25601859

  3. Advanced planning activity. [for interplanetary flight and space exploration

    NASA Technical Reports Server (NTRS)

    1974-01-01

    Selected mission concepts for interplanetary exploration through 1985 were examined, including: (1) Jupiter orbiter performance characteristics; (2) solar electric propulsion missions to Mercury, Venus, Neptune, and Uranus; (3) space shuttle planetary missions; (4) Pioneer entry probes to Saturn and Uranus; (5) rendezvous with Comet Kohoutek and Comet Encke; (6) space tug capabilities; and (7) a Pioneer mission to Mars in 1979. Mission options, limitations, and performance predictions are assessed, along with probable configurational, boost, and propulsion requirements.

  4. Activities of the Japanese space weather forecast center at Communications Research Laboratory.

    PubMed

    Watari, Shinichi; Tomita, Fumihiko

    2002-12-01

    The International Space Environment Service (ISES) is an international organization for space weather forecasts and belongs to the International Union of Radio Science (URSI). There are eleven ISES forecast centers in the world, and Communications Research Laboratory (CRL) runs the Japanese one. We make forecasts on the space environment and deliver them over the phones and through the Internet. Our forecasts could be useful for human activities in space. Currently solar activity is near maximum phase of the solar cycle 23. We report the several large disturbances of space environment occurred in 2001, during which low-latitude auroras were observed several times in Japan.

  5. Activities of the Space Studies Board of the National Research Council

    NASA Technical Reports Server (NTRS)

    1993-01-01

    This 1993 annual report of the Space Studies Board of the National Research Council chronicles the activities of the board during a year filled with questioning and change in the country's civil space program. The brief accounts contained herein of the activities of the board and of its committees, together with summaries of two major reports and the complete texts of three letter reports, sketch out major space research issues that faced the nation's space scientists and engineers during the year, including scientific prerequisites for the human exploration of space, improving NASA's technology for space science, the space station and prerequisites for the human exploration program, several issues in the space life sciences, and the Advanced X-ray Astrophysics Facility.

  6. Space Physics for Graduate Students: An Activities-Based Approach

    NASA Astrophysics Data System (ADS)

    Gross, N. A.; Arge, N.; Bruntz, R.; Burns, A. G.; Hughes, W. J.; Knipp, D.; Lyon, J.; McGregor, S.; Owens, M.; Siscoe, G.; Solomon, S. C.; Wiltberger, M.

    2009-01-01

    The geospace environment is controlled largely by events on the Sun, such as solar flares and coronal mass ejections, which generate significant geomagnetic and upper atmospheric disturbances. The study of this Sun-Earth system, which has become known as space weather, has both intrinsic scientific interest and practical applications. Adverse conditions in space can damage satellites and disrupt communications, navigation, and electric power grids, as well as endanger astronauts. The Center for Integrated Space Weather Modeling (CISM), a Science and Technology Center (STC) funded by the U.S. National Science Foundation (see http://www.bu.edu/cism/), is developing a suite of integrated physics-based computer models that describe the space environment from the Sun to the Earth for use in both research and operations [Hughes and Hudson, 2004, p. 1241]. To further this mission, advanced education and training programs sponsored by CISM encourage students to view space weather as a system that encompasses the Sun, the solar wind, the magnetosphere, and the ionosphere/thermosphere. This holds especially true for participants in the CISM space weather summer school [Simpson, 2004].

  7. Differences in associations between active transportation and built environmental exposures when expressed using different components of individual activity spaces.

    PubMed

    van Heeswijck, Torbjorn; Paquet, Catherine; Kestens, Yan; Thierry, Benoit; Morency, Catherine; Daniel, Mark

    2015-05-01

    This study assessed relationships between built environmental exposures measured within components of individual activity spaces (i.e., travel origins, destinations and paths in-between), and use of active transportation in a metropolitan setting. Individuals (n=37,165) were categorised as using active or sedentary transportation based on travel survey data. Generalised Estimating Equations analysis was used to test relationships with active transportation. Strength and significance of relationships between exposures and active transportation varied for different components of the activity space. Associations were strongest when including travel paths in expression of the built environment. Land use mix and greenness were negatively related to active transportation.

  8. Layer plate CAS assay for the quantitation of siderophore production and determination of exudation patterns for fungi.

    PubMed

    Andrews, Megan Y; Santelli, Cara M; Duckworth, Owen W

    2016-02-01

    The chrome azurol S (CAS) assay measures the chelating activity of siderophores, but its application (especially to fungi) is limited by toxicity issues. In this note, we describe a modified version of the CAS assay that is suitable for quantifying siderophore exudation for microorganisms, including fungi.

  9. Aeronautics and Space Report of the President: Fiscal Year 1998 Activities

    NASA Technical Reports Server (NTRS)

    1999-01-01

    The National Aeronautics and Space Act of 1958 directed the annual Aeronautics and Space Report to include a "comprehensive description of the programmed activities and the accomplishments of all agencies of the United States in the field of aeronautics and space activities during the preceding calendar year. In recent years, the reports have been prepared on a fiscal year (FY) basis, consistent with the budgetary period now used in programs of the Federal Government. This year's report covers activities that took place from October 1, 1997, through September 30, 1998. The activities of agencies included are NASA, the Department of Defense, The Federal Aviation Administration, the Department of Commerce, the Department of the Interior, the Federal Communications Commission, the Department of Agriculture, the National Science Foundation, the Department of State, the Department of Energy, the Smithsonian Institution, the Arms Control and Disarmament Agency, the Environmental Protection Agency, and the U.S. Information Agency. Appendices cover the U.S. Government Spacecraft Record, World Record of Space Launches Successful in Attaining Earth Orbit or Beyond , Successful Launches to Orbit on U.S. Launch Vehicles, October 1, 1997-September 30, 1998, U.S. and Russian Human Space Flights, 1961-September 30, 1998, U.S. Space Launch Vehicles, Space Activities of the U.S. Government-Historical Budget Summary, Space Activities of the U.S. Government-Budget Authority in Equivalent FY 1998 Dollars, Federal Space Activities Budget, Federal Aeronautics Budget, and a glossary

  10. The CRISPR-Cas system for plant genome editing: advances and opportunities.

    PubMed

    Kumar, Vinay; Jain, Mukesh

    2015-01-01

    Genome editing is an approach in which a specific target DNA sequence of the genome is altered by adding, removing, or replacing DNA bases. Artificially engineered hybrid enzymes, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), and the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) system are being used for genome editing in various organisms including plants. The CRISPR-Cas system has been developed most recently and seems to be more efficient and less time-consuming compared with ZFNs or TALENs. This system employs an RNA-guided nuclease, Cas9, to induce double-strand breaks. The Cas9-mediated breaks are repaired by cellular DNA repair mechanisms and mediate gene/genome modifications. Here, we provide a detailed overview of the CRISPR-Cas system and its adoption in different organisms, especially plants, for various applications. Important considerations and future opportunities for deployment of the CRISPR-Cas system in plants for numerous applications are also discussed. Recent investigations have revealed the implications of the CRISPR-Cas system as a promising tool for targeted genetic modifications in plants. This technology is likely to be more commonly adopted in plant functional genomics studies and crop improvement in the near future.

  11. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells

    PubMed Central

    Lee, Ciaran M; Cradick, Thomas J; Bao, Gang

    2016-01-01

    The clustered regularly-interspaced short palindromic repeats (CRISPR)—CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications. PMID:26782639

  12. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

    NASA Astrophysics Data System (ADS)

    Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

    2014-03-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

  13. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells.

    PubMed

    Lee, Ciaran M; Cradick, Thomas J; Bao, Gang

    2016-03-01

    The clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications.

  14. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

    PubMed

    Sternberg, Samuel H; Redding, Sy; Jinek, Martin; Greene, Eric C; Doudna, Jennifer A

    2014-03-06

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

  15. Conceptualizing and Comparing Neighborhood and Activity Space Measures for Food Environment Research

    PubMed Central

    Crawford, Thomas W.; Pitts, Stephanie B. Jilcott; McGuirt, Jared T.; Keyserling, Thomas C.; Ammerman, Alice S.

    2014-01-01

    Greater accessibility to geospatial technologies has led to a surge of spatialized public health research, much of which has focused on food environments. The purpose of this study was to analyze differing spatial measures of exposure to supermarkets and farmers’ markets among women of reproductive age in eastern North Carolina. Exposure measures were derived using participant-defined neighborhoods, investigator-defined road network neighborhoods, and activity spaces incorporating participants’ time space behaviors. Results showed that mean area for participant-defined neighborhoods (0.04 sq. miles) was much smaller than 2.0 mile road network neighborhoods (3.11 sq. miles) and activity spaces (26.36 sq. miles), and that activity spaces provided the greatest market exposure. The traditional residential neighborhood concept may not be particularly relevant for all places. Time-space approaches capturing activity space may be more relevant, particularly if integrated with mixed methods strategies. PMID:25306420

  16. Cas6 specificity and CRISPR RNA loading in a complex CRISPR-Cas system.

    PubMed

    Sokolowski, Richard D; Graham, Shirley; White, Malcolm F

    2014-06-01

    CRISPR-Cas is an adaptive prokaryotic immune system, providing protection against viruses and other mobile genetic elements. In type I and type III CRISPR-Cas systems, CRISPR RNA (crRNA) is generated by cleavage of a primary transcript by the Cas6 endonuclease and loaded into multisubunit surveillance/effector complexes, allowing homology-directed detection and cleavage of invading elements. Highly studied CRISPR-Cas systems such as those in Escherichia coli and Pseudomonas aeruginosa have a single Cas6 enzyme that is an integral subunit of the surveillance complex. By contrast, Sulfolobus solfataricus has a complex CRISPR-Cas system with three types of surveillance complexes (Cascade/type I-A, CSM/type III-A and CMR/type III-B), five Cas6 paralogues and two different CRISPR-repeat families (AB and CD). Here, we investigate the kinetic properties of two different Cas6 paralogues from S. solfataricus. The Cas6-1 subtype is specific for CD-family CRISPR repeats, generating crRNA by multiple turnover catalysis whilst Cas6-3 has a broader specificity and also processes a non-coding RNA with a CRISPR repeat-related sequence. Deep sequencing of crRNA in surveillance complexes reveals a biased distribution of spacers derived from AB and CD loci, suggesting functional coupling between Cas6 paralogues and their downstream effector complexes.

  17. CAS as Environments for Implementing Mathematical Microworlds.

    ERIC Educational Resources Information Center

    Alpers, Burkhard

    2002-01-01

    Investigates whether computer algebra systems (CAS) are suitable environments for implementing mathematical microworlds. Recalls what constitutes a microworld and explores how CAS can be used for implementation, stating potentials as well as limitations. Provides as an example the microworld "Formula 1", implemented in Maple Software. (Author/KHR)

  18. Configuration studies for active electrostatic space radiation shielding

    NASA Astrophysics Data System (ADS)

    Joshi, Ravindra P.; Qiu, Hao; Tripathi, Ram K.

    2013-07-01

    Developing successful and optimal solutions to mitigating the hazards of severe space radiation in deep space long duration missions is critical for the success of deep-space explorations. Space crews traveling aboard interplanetary spacecraft will be exposed to a constant flux of galactic cosmic rays (GCR), as well as intense fluxes of charged particles during solar particle events (SPEs). A recent report (Tripathi et al., Adv. Space Res. 42 (2008) 1043-1049), had explored the feasibility of using electrostatic shielding in concert with the state-of-the-art materials shielding technologies. Here we continue to extend the electrostatic shielding strategy and quantitatively examine a different configuration based on multiple toroidal rings. Our results show that SPE radiation can almost be eliminated by these electrostatic configurations. Also, penetration probabilities for novel structures such as toroidal rings are shown to be substantially reduced as compared to the simpler all-sphere geometries. More interestingly, the dimensions and aspect ratio of the toroidal rings could be altered and optimized to achieve an even higher degree of radiation protection.

  19. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum.

    PubMed

    Gao, Junping; Wang, Genhong; Ma, Sanyuan; Xie, Xiaodong; Wu, Xiangwei; Zhang, Xingtan; Wu, Yuqian; Zhao, Ping; Xia, Qingyou

    2015-01-01

    Genome editing is one of the most powerful tools for revealing gene function and improving crop plants. Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been used as a powerful and efficient tool for genome editing in various organisms. Here, we report genome editing in tobacco (Nicotiana tabacum) mediated by the CRISPR/Cas9 system. Two genes, NtPDS and NtPDR6, were used for targeted mutagenesis. First, we examined the transient genome editing activity of this system in tobacco protoplasts, insertion and deletion (indel) mutations were observed with frequencies of 16.2-20.3% after transfecting guide RNA (gRNA) and the nuclease Cas9 in tobacco protoplasts. The two genes were also mutated using multiplexing gRNA at a time. Additionally, targeted deletions and inversions of a 1.8-kb fragment between two target sites in the NtPDS locus were demonstrated, while indel mutations were also detected at both the sites. Second, we obtained transgenic tobacco plants with NtPDS and NtPDR6 mutations induced by Cas9/gRNA. The mutation percentage was 81.8% for NtPDS gRNA4 and 87.5% for NtPDR6 gRNA2. Obvious phenotypes were observed, etiolated leaves for the psd mutant and more branches for the pdr6 mutant, indicating that highly efficient biallelic mutations occurred in both transgenic lines. No significant off-target mutations were obtained. Our results show that the CRISPR/Cas9 system is a useful tool for targeted mutagenesis of the tobacco genome.

  20. Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation

    PubMed Central

    Dominguez, Antonia A.; Lim, Wendell A.; Qi, Lei S.

    2016-01-01

    The bacterial CRISPR–Cas9 system has emerged as a multifunctional platform for sequence-specific regulation of gene expression. This Review describes the development of technologies based on nuclease-deactivated Cas9, termed dCas9, for RNA-guided genomic transcription regulation, both by repression through CRISPR interference (CRISPRi) and by activation through CRISPR activation (CRISPRa). We highlight different uses in diverse organisms, including bacterial and eukaryotic cells, and summarize current applications of harnessing CRISPR–dCas9 for multiplexed, inducible gene regulation, genome-wide screens and cell fate engineering. We also provide a perspective on future developments of the technology and its applications in biomedical research and clinical studies. PMID:26670017

  1. 14 CFR 1266.102 - Cross-waiver of liability for agreements for activities related to the International Space Station.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... activities related to the International Space Station. 1266.102 Section 1266.102 Aeronautics and Space... liability for agreements for activities related to the International Space Station. (a) The objective of... exploration, exploitation, and use of outer space through the International Space Station (ISS). The...

  2. CAS SCF/CI calculations of potential energy surfaces of He 3+ and He 2+

    NASA Astrophysics Data System (ADS)

    Balasubramanian, K.; Liao, M. Z.; Lin, S. H.

    1987-12-01

    Complete active space MC SCF (CAS SCF) calculations followed by second-order configuration interaction (SOCI) calculations are carried out on the potential energy surfaces (bending surface, linear surfaces) of the 2Σ g+ ground state of He 3+. The potential minimum for the 2Σ g+ state occurs at a linear geometry with HeHe bond length of 1.248 Å. The binding energy of He 3+ with respect to He + He + + He was calculated to be 2.47 eV at the SOCI level. The energy required to dissociate He 3+ ( 2Σ g+) into He 2+ ( 2Σ u+) and He( 1S) is calculated to be 0.14 eV. The same level of SOCI calculations of He 2+ yield a De value of 2.36 eV.

  3. Minimizing Actuator-Induced Residual Error in Active Space Telescope Primary Mirrors

    DTIC Science & Technology

    2010-09-01

    Minimizing Actuator-Induced Residual Error in Active Space Telescope Primary Mirrors by Matthew William Smith Submitted to the Department of...4. TITLE AND SUBTITLE Minimizing Actuator-Induced Residual Error in Active Space Telescope Primary Mirrors 5a. CONTRACT NUMBER 5b. GRANT NUMBER...release; distribution unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Heritage space telescope mirror technology-i.e. large, monolithic glass primary

  4. A priori complete active space self consistent field localized orbitals: an application on linear polyenes

    NASA Astrophysics Data System (ADS)

    Angeli, Celestino; Sparta, Manuel; Cimiraglia, Renzo

    2006-03-01

    A recently proposed a priori localization technique is used to exploit the possibility to reduce the number of active orbitals in a Complete Active Space Self Consistent Field calculation. The work relies on the fact that the new approach allows a strict control on the nature of the active orbitals and therefore makes it possible to include in the active space only the relevant orbitals. The idea is tested on the calculation of the energy barrier for rigid rotation of linear polyenes. In order to obtain a relevant set of data, a number of possible rotations around double bonds have been considered in the ethylene, butadiene, hexatriene, octatetraene, decapentaene, dodecahexaene molecules. The possibility to reduce the dimension of the active space has been investigated, considering for each possible rotation different active spaces ranging from the minimal dimension of 2 electrons in 2 π orbitals to the π-complete space. The results show that the rigid isomerization in the polyene molecules can be described with a negligible loss in accuracy with active spaces no larger than ten orbitals and ten electrons. In the special case of the rotation around the terminal double bond, the space can be further reduced to six orbitals and six electrons with a large decrease of the computational cost. An interesting summation rule has been found and verified for the stabilization of the energy barriers as a function of the dimension of the conjugated lateral chains and of the dimension of the active space.

  5. Earth-Space Science Activity Syllabus for Elementary and Junior High School Teachers of Science.

    ERIC Educational Resources Information Center

    Maier, Jack; And Others

    This syllabus is a collection of earth-space science laboratory activities and demonstrations intended for use at the elementary and junior high school levels. The activities are grouped into eight subject sections: Astronomy, Light, Magnetism, Electricity, Geology, Weather, Sound, and Space. Each section begins with brief background information,…

  6. CAS

    SciTech Connect

    Martinez, B.; Pomeroy, G. )

    1989-12-02

    The Security Alarm System is a data acquisition and control system which collects data from intrusion sensors and displays the information in a real-time environment for operators. The Access Control System monitors and controls the movement of personnel with the use of card readers and biometrics hand readers.

  7. Space Resources for Teachers, Space Science, A Guide Outlining Understandings, Fundamental Concepts, and Activities.

    ERIC Educational Resources Information Center

    Thompson, Malcolm

    This instructional and resource guide is designed so that it may be used in the secondary school or in the first two years of college to present a series of units in space science, or to supplement existing science and mathematics courses. The guide consists of six units: (1) measurement, distance, and size in astronomy, (2) atoms, spectra, and…

  8. Looking at Earth from Space: Teacher's Guide with Activities for Earth and Space Science.

    ERIC Educational Resources Information Center

    National Aeronautics and Space Administration, Washington, DC.

    The Maryland Pilot Earth Science and Technology Education Network (MAPS-NET) project was sponsored by the National Aeronautics and Space Administration (NASA) to enrich teacher preparation and classroom learning in the area of Earth system science. This publication includes a teacher's guide that replicates material taught during a graduate-level…

  9. Design and implementation of active members for precision space structures

    NASA Technical Reports Server (NTRS)

    Webster, M. S.; Fanson, J. L.; Lurie, B. J.; O'Brien, J. F.

    1992-01-01

    This paper describes the development and implementation of an active member in a precision truss structure. The active member utilizes a piezoelectric actuator motor imbedded in a steel case with built-in displacement sensor. This active member is used in structural quieting. Collocated active damping control loops are designed in order to impedance match piezoelectric active members to the structure. Results from application of these controllers and actuators to the JPL Phase B testbed are given.

  10. The places parents go: understanding the breadth, scope, and experiences of activity spaces for parents.

    PubMed

    Wolf, Jennifer Price; Freisthler, Bridget; Kepple, Nancy Jo; Chávez, Raúl

    2017-04-01

    Neighborhood environments are related to parenting behaviors, which in turn have a life-long effect on children's health and well-being. Activity spaces, which measure individual routine patterns of movement, may be helpful in assessing how physical and social environments shape parenting. In this study we use qualitative data and GIS mapping from 4 California cities to examine parental activity spaces. Parents described a number of factors that shape their activity spaces including caregiving status, the age of their children, and income. Parental activity spaces also varied between times (weekends vs. weekdays) and places (adult-only vs. child-specific places). Knowing how to best capture and study parental activity spaces could identify mechanisms by which environmental factors influence parenting behaviors and child health.

  11. The places parents go: understanding the breadth, scope, and experiences of activity spaces for parents

    PubMed Central

    Wolf, Jennifer Price; Freisthler, Bridget; Kepple, Nancy Jo; Chávez, Raúl

    2016-01-01

    Neighborhood environments are related to parenting behaviors, which in turn have a life-long effect on children’s health and well-being. Activity spaces, which measure individual routine patterns of movement, may be helpful in assessing how physical and social environments shape parenting. In this study we use qualitative data and GIS mapping from 4 California cities to examine parental activity spaces. Parents described a number of factors that shape their activity spaces including caregiving status, the age of their children, and income. Parental activity spaces also varied between times (weekends vs. weekdays) and places (adult-only vs. child-specific places). Knowing how to best capture and study parental activity spaces could identify mechanisms by which environmental factors influence parenting behaviors and child health. PMID:28392621

  12. Comparative assessments of CRISPR-Cas nucleases' cleavage efficiency in planta.

    PubMed

    Johnson, Ross A; Gurevich, Vyacheslav; Filler, Shdema; Samach, Aviva; Levy, Avraham A

    2015-01-01

    Custom-designed nucleases can enable precise plant genome editing by catalyzing DNA-breakage at specific targets to stimulate targeted mutagenesis or gene replacement. The CRISPR-Cas system, with its target-specifying RNA molecule to direct the Cas9 nuclease, is a recent addition to existing nucleases that bind and cleave the target through linked protein domains (e.g. TALENs and zinc-finger nucleases). We have conducted a comparative study of these different types of custom-designed nucleases and we have assessed various components of the CRISPR-Cas system. For this purpose, we have adapted our previously reported assay for cleavage-dependent luciferase gene correction in Nicotiana benthamiana leaves (Johnson et al. in Plant Mol Biol 82(3):207-221, 2013). We found that cleavage by CRISPR-Cas was more efficient than cleavage of the same target by TALENs. We also compared the cleavage efficiency of the Streptococcus pyogenes Cas9 protein based on expression using three different Cas9 gene variants. We found significant differences in cleavage efficiency between these variants, with human and Arabidopsis thaliana codon-optimized genes having the highest cleavage efficiencies. We compared the activity of 12 de novo-designed single synthetic guide RNA (sgRNA) constructs, and found their cleavage efficiency varied drastically when using the same Cas9 nuclease. Finally, we show that, for one of the targets tested with our assay, we could induce a germinally-transmitted deletion in a repeat array in A. thaliana. This work emphasizes the efficiency of the CRISPR-Cas system in plants. It also shows that further work is needed to be able to predict the optimal design of sgRNAs or Cas9 variants.

  13. Activity of Science and Operational Research of NICT Space Weather

    NASA Astrophysics Data System (ADS)

    Ishii, Mamoru; Nagatsuma, Tsutomu; Watari, Shinichi; Shinagawa, Hiroyuki; Tsugawa, Takuya; Kubo, Yuki

    Operational space weather forecast is for contribution to social infrastructure than for academic interests. These user need will determine the target of research, e.g., the precision level, spatial and temporal resolution and/or required lead time. We, NICT, aim two target in the present mid-term strategic plan, which are (1) forecast of ionospheric disturbance influencing to satellite positioning, and (2) forecast of disturbance in radiation belt influencing to satellite operation. We have our own observation network and develop empirical and numerical models for achieving each target. However in actual situation, it is much difficult to know the user needs quantitatively. Most of space weather phenomena makes the performance of social infrastructure poor, for example disconnect of HF communication, increase of GNSS error. Most of organizations related to these operation are negative to open these information. We have personal interviews to solve this issue. In this interview, we try to collect incident information related to space weather in each field, and to retrieve which space weather information is necessary for users. In this presentation we will introduce our research and corresponding new service, in addition to our recent scientific results.

  14. Conscientization and Third Space: A Case Study of Tunisian Activism

    ERIC Educational Resources Information Center

    Boumlik, Habiba; Schwartz, Joni

    2016-01-01

    This case study examines, "Al Bawsala," a nongovernmental organization and a female cyber social activist, Amira Yahyaoui, in the aftermath of Tunisia's Jasmine Revolution through the lens of adult education. The theoretical frameworks of conscientization and third space are employed to describe Yahyaoui's development of the watchdog…

  15. Search and Rescue in Space Activities: Is There a Specific Legal Regime?

    NASA Astrophysics Data System (ADS)

    Kyriakopoulos, George D.

    2013-09-01

    As space exploration has always been a dangerous activity, this paper examines if a "space" search and rescue regime can validly be claimed to exist. Regarding vessels in distress, the Hamburg Convention provided for SAR regions "by way of mutual regional arrangements" between States. Regarding aircraft in distress, specific SAR Regions have been established, in conformity with Annex 12 to the Chicago Convention. In space, art. V of the OST, if broadly interpreted, could be applied to "space-to space" rescue operations. This principle is further elaborated by the Rescue Agreement, although the latter does not apply in "deep space" distress situations. Finally, The Moon Agreement applies this framework to "Persons on the Moon". It follows that Space Law provides for SAR operations on the level of principle, as no integrated legal regime seems to exist. Therefore, the Air/Sea SAR regimes could serve as models for a comprehensive space regulation.

  16. Effects of Crude Oil/Dispersant Mixture and Dispersant Components on PPARγ Activity in Vitro and in Vivo: Identification of Dioctyl Sodium Sulfosuccinate (DOSS; CAS #577-11-7) as a Probable Obesogen

    PubMed Central

    Temkin, Alexis M.; Bowers, Robert R.; Magaletta, Margaret E.; Holshouser, Steven; Maggi, Adriana; Ciana, Paolo; Guillette, Louis J.; Bowden, John A.; Kucklick, John R.; Baatz, John E.; Spyropoulos, Demetri D.

    2015-01-01

    adipocyte differentiation. Conclusions We conclude that DOSS is a putative obesogen worthy of further study, including epidemiological and clinical investigations into laxative prescriptions consisting of DOSS. Citation Temkin AM, Bowers RR, Magaletta ME, Holshouser S, Maggi A, Ciana P, Guillette LJ, Bowden JA, Kucklick JR, Baatz JE, Spyropoulos DD. 2016. Effects of crude oil/dispersant mixture and dispersant components on PPARγ activity in vitro and in vivo: identification of dioctyl sodium sulfosuccinate (DOSS; CAS #577-11-7) as a probable obesogen. Environ Health Perspect 124:112–119; http://dx.doi.org/10.1289/ehp.1409672 PMID:26135921

  17. Using the CRISPR-Cas System to Positively Select Mutants in Genes Essential for Its Function.

    PubMed

    Yosef, Ido; Goren, Moran G; Edgar, Rotem; Qimron, Udi

    2015-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) comprise a prokaryotic adaptive defense system against foreign nucleic acids. This defense is mediated by Cas proteins, which are guided by sequences flanked by the repeats, called spacers, to target nucleic acids. Spacers designed against the prokaryotic self chromosome are lethal to the prokaryotic cell. This self-killing of the bacterium by its own CRISPR-Cas system can be used to positively select genes that participate in this killing, as their absence will result in viable cells. Here we describe a positive selection assay that uses this feature to identify E. coli mutants encoding an inactive CRISPR-Cas system. The procedure includes establishment of an assay that detects this self-killing, generation of transposon insertion mutants in random genes, and selection of viable mutants, suspected as required for this lethal activity. This procedure enabled us to identify a novel gene, htpG, that is required for the activity of the CRISPR-Cas system. The procedures described here can be adjusted to various organisms to identify genes required for their CRISPR-Cas activity.

  18. Does activity space size influence physical activity levels of adolescents?—A GPS study of an urban environment

    PubMed Central

    Lee, Nolan C.; Voss, Christine; Frazer, Amanda D.; Hirsch, Jana A.; McKay, Heather A.; Winters, Meghan

    2015-01-01

    Background Physical activity (PA) is closely linked with child and youth health, and active travel may be a solution to enhancing PA levels. Activity spaces depict the geographic coverage of one's travel. Little is known about activity spaces and PA in adolescents. Objective To explore the relation between adolescent travel (using a spatial measure of activity space size) and daily moderate-to-vigorous PA (MVPA), with a focus on school days. Methods We used Global Positioning Systems to manually identify trips and generate activity spaces for each person-day; quantified by area for 39 students (13.8 ± 0.6 years, 38% female) attending high school in urban Downtown Vancouver, Canada. We assessed the association between activity space area and MVPA using multi-level regression. We calculated total, school-day and trip-based MVPA for each valid person-day (accelerometry; ≥ 600 min wear time). Results On school days, students accrued 68.2 min/day (95% CI 60.4–76.0) of MVPA. Daily activity spaces averaged 2.2 km2 (95% CI 1.3–3.0). There was no association between activity space size and school-day MVPA. Students accrued 21.8 min/day (95% CI 19.2–24.4) of MVPA during school hours, 19.4 min/day (95% CI 15.1–23.7) during travel, and 28.3 min/day (95% CI 22.3–34.3) elsewhere. Conclusion School and school travel are important sources of PA in Vancouver adolescents, irrespective of activity space area covered. PMID:26807349

  19. Report on Advanced Life Support Activities at Kennedy Space Center

    NASA Technical Reports Server (NTRS)

    Wheeler, Raymond M.

    2004-01-01

    Plant studies at Kennedy Space Center last year focused on selecting cultivars of lettuce, tomato, and pepper for further testing as crops for near-term space flight applications. Other testing continued with lettuce, onion, and radish plants grown at different combinations of light (PPF), temperature, and CO2 concentration. In addition, comparisons of mixed versus mono culture approaches for vegetable production were studied. Water processing testing focused on the development and testing of a rotating membrane bioreactor to increase oxygen diffusion levels for reducing total organic carbon levels and promoting nitrification. Other testing continued to study composting testing for food wastes (NRA grant) and the use of supplemental green light with red/blue LED lighting systems for plant production (NRC fellowship).

  20. Russian Activities in Space Photovoltaic Power Modules with Concentrators

    NASA Technical Reports Server (NTRS)

    Andreev, Vyacheslav M.; Rumyantsev, Valeri D.

    2004-01-01

    Space concentrator modules with point-and line-focus Fresnel lenses and with reflective parabolic troughs have been developed recently at Ioffe Physico-Technical Institute. PV receivers for these modules are based: on the single junction LPE and MOCVD AlGaAs/GaAs solar cells characterized by AM0 efficiencies of 23.5 - 24% at 20 - 50 suns and 24 - 24.75 at 50 - 200 suns; on the mechanically stacked tandem AlGaAs/GaAs-GaSb cells with efficiency of 27 - 28 at 20 - 100 suns. MOCVD AlGaAs/GaAs cells with internal Bragg reflector have shown a higher radiation resistance as compared to a traditional structure. Monolithic two-terminal tandems AlGaAs (top)-GaAs (bottom) for space application and GaSb (top) - InGaAsSb (bottom) for TRV application are under development as well.

  1. Current and Projected Government and Commercial Space Activities

    DTIC Science & Technology

    1975-04-01

    satellites. Reimbursable. Figure 1 Space Launches by NASA in 197 5—Continued m mm* m~ammm^m i^» ■^ Mission Improved TIPOS Operational Satellite...expects to be doing $12 billion in annual business. An additional market of $2 billion for viral insecticides for the year 2000 also is forecast...terminals, and collecting data utilizing thousands of small terrestrial telecommunications units. The ATS-F is expected to create new markets in

  2. Aerospace Battery Activities at NASA/Goddard Space Flight Center

    NASA Technical Reports Server (NTRS)

    Rao, Gopalakrishna M.

    2006-01-01

    Goddard Space Flight Center has "pioneered" rechargeable secondary battery design, test, infusion and in-orbit battery management among NASA installations. Nickel cadmium batteries of various designs and sizes have been infused for LEO, GEO and Libration Point spacecraft. Nickel-Hydrogen batteries have currently been baselined for the majority of our missions. Li-Ion batteries from ABSL, JSB, SaFT and Lithion have been designed and tested for aerospace application.

  3. Recent and Future Stratospheric Balloon Activities at Esrange Space Center

    NASA Astrophysics Data System (ADS)

    Kemi, Stig

    Esrange Space Center located in northern Sweden has during 45 years been a leading launch site for both sounding rockets and stratospheric balloons. We have a unique combination of maintaining both stratospheric balloons and sounding rockets launch operations. Most balloon flights are normally handled inside Scandinavia but since 2005 PersonNamesemi-circular flights are performed with recovery in northern Canada. The Swedish Government and Swedish National Space Board are now finaliz-ing an agreement with Russia for peaceful uPersonNamese of space, which will permit circumpolar balloon flights. Within this agreement we will soon be able to of-fer the science community long duration balloon flights with durations for PersonNameseveral weeks. The balloon operations at Esrange Space Center are yearly expanding. Both NASA and CNES have long term plans for balloon flights from northern Sweden. We have also received a request from JAXA for future balloon missions. To handle balloon campaigns with large numbers of payloads or build up for two different campaigns a new big assembly hall will be ready for use at the beginning of 2011. January 24 we made an historical balloon flight in a very cold stratosphere with a Zodiac metricconverterProductID402?000 m3402ü ınbsp;000 m3402 000 m3 balloon carrying a 750kg gondola with the German Mipas-B/Telis instrument. The balloon reached 34kms alti-tude after a carefully piloted ascent in temperature levels down to -89 degrees Centigrade. The scientists received unique data during the 13 hours and 30 minutes long sailing at different altitudes during slow descent. The payload was recovered in very good condition 80 kms from the border between country-regionFinland and Russia.

  4. Reliability issues in active control of large flexible space structures

    NASA Technical Reports Server (NTRS)

    Vandervelde, W. E.

    1983-01-01

    The unreliability of control system components was investigated in our attempt to deal with that problem. This matter is of concern in large space structure control because of the large number of components required to achieve specified performance in some situations, and the long operating period required between maintenance visits. The detection and isolation of component failures during system operation, and algorithms for reconfiguring control systems following detection and isolation of a failure were emphasized.

  5. RXTE Observations of Cas A

    NASA Technical Reports Server (NTRS)

    Rothschild, R. E.; Lingenfelter, R. E.; Heindl, W. A.; Blanco, P. R.; Pelling, M. R.; Gruber, D. E.; Allen, G. E.; Jahoda, K.; Swank, J. H.; Woosley, S. E.; Nomoto, K.; Higdon, J. C.; Dermer, Charles D. (Editor); Strickman, Mark S. (Editor); Kurfess, James D. (Editor)

    1997-01-01

    The exciting detection by the COMPTEL instrument of the 1157 keV Ti-44 line from the supernova remnant Cas A sets important new constraints on supernova dynamics and nucleosynthesis. The Ti-44 decay also produces x-ray lines at 68 and 78 keV, whose flux should be essentially the same as that of the gamma ray line. The revised COMPTEL flux of 4 x l0(exp -5) cm(exp -2)s(exp -1) is very near the sensitivity limit for line detection by the HEXTE instrument on RXTE. We report on the results from two RXTE observations - 20 ks during In Orbit Checkout in January 1996 and 200 ks in April 1996. We also find a strong continuum emission suggesting cosmic ray electron acceleration in the remnant.

  6. Genome Editing in Escherichia coli with Cas9 and synthetic CRISPRs

    SciTech Connect

    Peng, Ze; Richardson, Sarah; Robinson, David; Deutsch, Samuel; Cheng, Jan-Fang

    2014-03-14

    Recently, the Cas9-CRISPR system has proven to be a useful tool for genome editing in eukaryotes, which repair the double stranded breaks made by Cas9 with non-homologous end joining or homologous recombination. Escherichia coli lacks non-homologous end joining and has a very low homologous recombination rate, effectively rendering targeted Cas9 activity lethal. We have developed a heat curable, serializable, plasmid based system for selectionless Cas9 editing in arbitrary E. coli strains that uses synthetic CRISPRs for targeting and -red to effect repairs of double stranded breaks. We have demonstrated insertions, substitutions, and multi-target deletions with our system, which we have tested in several strains.

  7. CRISPR/Cas9: an advanced tool for editing plant genomes.

    PubMed

    Samanta, Milan Kumar; Dey, Avishek; Gayen, Srimonta

    2016-10-01

    To meet current challenges in agriculture, genome editing using sequence-specific nucleases (SSNs) is a powerful tool for basic and applied plant biology research. Here, we describe the principle and application of available genome editing tools, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat associated CRISPR/Cas9 system. Among these SSNs, CRISPR/Cas9 is the most recently characterized and rapidly developing genome editing technology, and has been successfully utilized in a wide variety of organisms. This review specifically illustrates the power of CRISPR/Cas9 as a tool for plant genome engineering, and describes the strengths and weaknesses of the CRISPR/Cas9 technology compared to two well-established genome editing tools, ZFNs and TALENs.

  8. Application of the French Space Operation Act and the Development of Space Activities in the Field of Launchers

    NASA Astrophysics Data System (ADS)

    Cahuzac, F.; Biard, A.

    2012-01-01

    The development of space activities has led France to define a new legal framework: French Space Operation Act (FSOA). The aim of this act, is to define the conditions according to which the French government authorizes and checks the spatial operations under its jurisdiction or its international responsibility as State of launch, according to the international treaties of the UN on space, in particular the Treaty (1967) on Principles Governing the Activities of States in the Exploration and Use of Outer Space, the Convention ( 1972 ) on International Liability for Damage Caused by Space Objects, and the Convention (1975) on Registration of Objects Launched into Outer Space. The main European space centre is the Guiana Space Centre (CSG), settled in France. A clarification of the French legal framework was compulsory to allow the arrival of new launchers (Soyuz and Vega). This act defines the competent authority, the procedure of authorization and licenses, the regime for operations led from foreign countries, the control of spatial objects, the enabling of inspectors, the delegation of monitoring to CNES, the procedure for urgent measures necessary for the safety, the registration of spatial objects. In this framework, the operator is fully responsible of the operation that he leads. He is subjected to a regime of authorization and to governmental technical monitoring delegated to CNES. In case of litigation, the operator gets the State guarantee above a certain level of damage to third party. The introduction of FSOA has led to issue a Technical Regulation set forth, in particular for the safety of persons and property, the protection of public health and the environment. This general regulation is completed by a specific regulation applicable to CSG that covers the preparation phase of the launch, and all specificities of the launch range, as regards the beginning of the launch. The Technical Regulation is based on 30 years of Ariane's activities and on the

  9. Republic of Kazakhstan: Capacity Building through the Increasing of Space Activities

    NASA Astrophysics Data System (ADS)

    Omarova, G.

    Currently, a new space policy of the Republic of Kazakhstan is being formulated. Basic directions are: Adherence to principal agreements of the International Space Law. Optimal utilization and modernization of the Baikonur spaceport launch infrastructure. Creation of the national satellite communication system In accordance with the above listed goals and objectives, the following priority actions should be taken in national level: Increasing of the National activities in COPUOS Developing of the National space activities Program and Space activities Act; Funding of a new and upgraded facilities at the Baikonur spaceport; Creating of the educational and training system for national space industry In 2004 Kazakhstan-Russia cooperation in space activities has entered to a new perspectives. Both countries proceeded to develop joint projects in the field of space activities connected to modernization of existing space infrastructure of the Baikonur spaceport for launchers that meet requirements of ecological security. Three relevant bilateral agreements were signed. All signed documents ensure more wide participation of the Republic of Kazakhstan in realization of space programs and projects implemented at the Baikonur spaceport through shared financing and realization jointly with Russia of projects on building of the space missile complex ``Baiterek'' and launching of geostationary communication satellite. It opens great opportunities for Kazakhstan in terms of capacity building. Implementation of the mentioned two projects will allow to use the available scientific, technical and intellectual potential of the Republic of Kazakhstan in the field of space activities, and to utilize effectively the infrastructure of Baikonur complex, to get affordable access to space technologies, to create conditions for development, test and operation of space facilities, new science --capacity technologies that will lead to close integration with Russian space industry and with

  10. Collaboration with aviation — The key to commercialisation of space activities

    NASA Astrophysics Data System (ADS)

    Collins, Patrick; Funatsu, Yoshiyuki

    2000-07-01

    The US government's Commercial Space Act of 1998 and commitment to commercialise the International Space Station's operations have changed the direction of space development in the post-cold-war world definitively. During 1998 also the feasibility and great economic potential of space travel by the general public was acknowledged in publications by NASA, AIAA and the Japanese Keidanren. However, crewed space activities are all taxpayer-funded, primarily for scientific research; they have involved only a few hundred people traveling to space to date; and those involved have no experience of commercial passenger service operations. By contrast, aviation is a global industry, largely commercial, involving the range of activities from engineering design to marketing, and serving more than 1 billion passengers/year. Aviation has very high safety levels developed over decades of experience of carrying billions of passengers. Furthermore, the aviation industry also has extensive experience of operating rocket-powered piloted vehicles: during the 1950s several countries operated such vehicles sufficiently frequently to develop routine operations, maintenance and repair procedures. Consequently, in order to develop safe and profitable passenger travel services to, from and in space, people, companies and organisations with experience of space activities have a great deal to gain from collaboration with all parts of the aviation industry. Due to the potential economic value of this development, and the high cost to taxpayers of space activities today, governments should take steps to start this collaboration as soon as possible.

  11. Effects of habitat and urbanization on the active space of brown-headed cowbird song.

    PubMed

    Gall, Megan D; Ronald, Kelly L; Bestrom, Eric S; Lucas, Jeffrey R

    2012-12-01

    The ability of a receiver to detect a signal is a product of the signal characteristics at the sender, habitat-specific degradation of the signal, and properties of the receiver's sensory system. Active space describes the maximum distance at which a receiver with a given sensory system can detect a signal in a given habitat. Here the effect of habitat structure and urbanization on brown-headed cowbird (Molothrus ater) perched song active space was explored. The active space of the cowbird song was affected by both habitat type and level of urbanization. High frequency (4 to 6 kHz) portions of song resulted in the maximum active space. Surprisingly, the active space was the largest in open urban environments. The hard surfaces found in open urban areas (e.g., sidewalks, buildings) may provide a sound channel that enhances song propagation. When the introductory phrase and final phrase were analyzed separately, the active space of the introductory phrase was found to decrease in open urban environments but the active space of the final phrase increased in open urban environments. This suggests that different portions of the vocalization may be differentially influenced by habitat and level of urbanization.

  12. Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

    PubMed Central

    Zuo, Zhicheng; Liu, Jin

    2016-01-01

    The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends. PMID:27874072

  13. Cullin 5 destabilizes Cas to inhibit Src-dependent cell transformation.

    PubMed

    Teckchandani, Anjali; Laszlo, George S; Simó, Sergi; Shah, Khyati; Pilling, Carissa; Strait, Alexander A; Cooper, Jonathan A

    2014-02-01

    Phosphorylation-dependent protein ubiquitylation and degradation provides an irreversible mechanism to terminate protein kinase signaling. Here, we report that mammary epithelial cells require cullin-5-RING-E3-ubiquitin-ligase complexes (Cul5-CRLs) to prevent transformation by a Src-Cas signaling pathway. Removal of Cul5 stimulates growth-factor-independent growth and migration, membrane dynamics and colony dysmorphogenesis, which are all dependent on the endogenous tyrosine kinase Src. Src is activated in Cul5-deficient cells, but Src activation alone is not sufficient to cause transformation. We found that Cul5 and Src together stimulate degradation of the Src substrate p130Cas (Crk-associated substrate). Phosphorylation stimulates Cas binding to the Cul5-CRL adaptor protein SOCS6 and consequent proteasome-dependent degradation. Cas is necessary for the transformation of Cul5-deficient cells. Either knockdown of SOCS6 or use of a degradation-resistant Cas mutant stimulates membrane ruffling, but not other aspects of transformation. Our results show that endogenous Cul5 suppresses epithelial cell transformation by several pathways, including inhibition of Src-Cas-induced ruffling through SOCS6.

  14. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes

    SciTech Connect

    Carte, Jason; Wang, Ruiying; Li, Hong; Terns, Rebecca M.; Terns, Michael P.

    2010-11-09

    An RNA-based gene silencing pathway that protects bacteria and archaea from viruses and other genome invaders is hypothesized to arise from guide RNAs encoded by CRISPR loci and proteins encoded by the cas genes. CRISPR loci contain multiple short invader-derived sequences separated by short repeats. The presence of virus-specific sequences within CRISPR loci of prokaryotic genomes confers resistance against corresponding viruses. The CRISPR loci are transcribed as long RNAs that must be processed to smaller guide RNAs. Here we identified Pyrococcus furiosus Cas6 as a novel endoribonuclease that cleaves CRISPR RNAs within the repeat sequences to release individual invader targeting RNAs. Cas6 interacts with a specific sequence motif in the 5{prime} region of the CRISPR repeat element and cleaves at a defined site within the 3{prime} region of the repeat. The 1.8 angstrom crystal structure of the enzyme reveals two ferredoxin-like folds that are also found in other RNA-binding proteins. The predicted active site of the enzyme is similar to that of tRNA splicing endonucleases, and concordantly, Cas6 activity is metal-independent. cas6 is one of the most widely distributed CRISPR-associated genes. Our findings indicate that Cas6 functions in the generation of CRISPR-derived guide RNAs in numerous bacteria and archaea.

  15. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

    PubMed

    Kabadi, Ami M; Ousterout, David G; Hilton, Isaac B; Gersbach, Charles A

    2014-10-29

    Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

  16. Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

    NASA Astrophysics Data System (ADS)

    Zuo, Zhicheng; Liu, Jin

    2016-11-01

    The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

  17. The therapeutic application of CRISPR/Cas9 technologies for HIV

    PubMed Central

    Saayman, Sheena; Ali, Stuart A.; Morris, Kevin V.; Weinberg, Marc S.

    2015-01-01

    Introduction The use of antiretroviral therapy (ART) has led to a significant decrease in morbidity and mortality in HIV-infected individuals. Nevertheless gene-based therapies represent a promising therapeutic paradigm for HIV-1, as they have the potential for sustained viral inhibition and reduced treatment interventions. One new method amendable to a gene-based therapy is the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing system. Areas covered CRISPR/Cas9 can be engineered to successfully modulate an array of disease-causing genetic elements. We discuss the diverse roles that CRISPR/Cas9 may play in targeting HIV and eradicating infection. The Cas9 nuclease coupled with one or more small guide RNAs (sgRNAs) can target the provirus to mediate excision of the integrated viral genome. Moreover, a modified nuclease deficient Cas9 fused to transcription activating domains may induce targeted activation of proviral gene expression allowing for the purging of the latent reservoirs. These technologies can also be exploited to target host dependency factors such as the co-receptor CCR5, thus preventing cellular entry of the virus. Expert opinion The diversity of the CRISPR/Cas9 technologies hold great promise for targeting different stages of the viral life cycle, and have the capacity for mediating an effective and sustained genetic therapy against HIV. PMID:25865334

  18. Radiological health risks to astronauts from space activities and medical procedures

    NASA Technical Reports Server (NTRS)

    Peterson, Leif E.; Nachtwey, D. Stuart

    1990-01-01

    Radiation protection standards for space activities differ substantially from those applied to terrestrial working situations. The levels of radiation and subsequent hazards to which space workers are exposed are quite unlike anything found on Earth. The new more highly refined system of risk management involves assessing the risk to each space worker from all sources of radiation (occupational and non-occupational) at the organ level. The risk coefficients were applied to previous space and medical exposures (diagnostic x ray and nuclear medicine procedures) in order to estimate the radiation-induced lifetime cancer incidence and mortality risk. At present, the risk from medical procedures when compared to space activities is 14 times higher for cancer incidence and 13 times higher for cancer mortality; however, this will change as the per capita dose during Space Station Freedom and interplanetary missions increases and more is known about the risks from exposure to high-LET radiation.

  19. Radiological health risks to astronauts from space activities and medical procedures

    SciTech Connect

    Paterson, L.E.; Nachtwey, D.S.

    1990-08-01

    Radiation protection standards for space activities differ substantially from those applied to terrestrial working situations. The levels of radiation and subsequent hazards to which space workers are exposed are quite unlike anything found on Earth. The new more highly refined system of risk management involves assessing the risk to each space worker from all sources of radiation (occupational and non-occupational) at the organ level. The risk coefficients were applied to previous space and medical exposures (diagnostic x ray and nuclear medicine procedures) in order to estimate the radiation-induced lifetime cancer incidence and mortality risk. At present, the risk from medical procedures when compared to space activities is 14 times higher for cancer incidence and 13 times higher for cancer mortality; however, this will change as the per capita dose during Space Station Freedom and interplanetary missions increases and more is known about the risks from exposure to high-LET radiation.

  20. Recent and future Stratospheric Balloon Activities at Esrange Space Center

    NASA Astrophysics Data System (ADS)

    Kemi, Stig

    2012-07-01

    PlaceNameEsrange PlaceNameSpace PlaceTypeCenter located in northern country-regionplaceSweden has during 45 years been a leading launch site for both sounding rockets and stratospheric balloons. We have an unique combination of maintaining both stratospheric balloons and sounding rockets launch operations. Most balloon flights are normally handled inside Scandinavia but since 2005 PersonNamesemi-circular flights are performed with recovery in northern country-regionplaceCanada. The Swedish and Russian Governments have signed an agreement for peaceful exploration of space on 19 March 2010, which will permit circumpolar balloon flights. Within this agreement we are able to offer the science community long duration balloon flights in the Northern Hemisphere with durations for PersonNameseveral weeks. The balloon operations at placePlaceNameEsrange PlaceNameSpace PlaceTypeCenter are yearly expanding. Both NASA and CNES have long term plans for balloon flights from northern country-regionplaceSweden. We have also received requests from placePlaceNameJapanese PlaceTypeUniversities and JAXA for future balloon missions. To handle balloon campaigns with large numbers of payloads or build up for two different campaigns a new big assembly hall was ready for use in April 2011. Circumpolar balloon flights from PlaceNameplaceEsrange PlaceNameSpace PlaceTypeCenter are possible due to the specific conditions during the Arctic summer with continuous daylight and nearly constant solar heating keeping the balloon at a constant altitude with a minimum of ballast. In total 10 payloads have been flying for 4 to 5 days from Esrange westwards with landing in northern Canada since 2005. The SUNRISE balloon borne solar telescope is one example which made in June metricconverterProductID2009 a2009 a more than 4 days semi-circular balloon flight from Esrange. The CitySunrise project is a collaborative project between the Max Planck Institute for Solar System Research in Katlenburg-Lindau and

  1. Aeronautics and Space Report of the President: Fiscal Year 2007 Activities

    NASA Technical Reports Server (NTRS)

    2009-01-01

    The National Aeronautics and Space Act of 1958 directed the annual Aeronautics and Space Report to include a "comprehensive description of the programmed activities and the accomplishments of all agencies of the United States in the field of aeronautics and space activities during the preceding calendar year." In recent years, the reports have been prepared on a fiscal-year basis, consistent with the budgetary period now used in programs of the Federal Government. This year's report covers activities that took place from October 1, 2006, through September 30, 2007.

  2. Aeronautics and Space Report of the President: Fiscal Year 1999 Activities

    NASA Technical Reports Server (NTRS)

    1999-01-01

    The National Aeronautics and Space Act of 1958 directed the annual Aeronautics and Space Report to include a "comprehensive description of the programmed activities and the accomplishments of all agencies of the United States in the field of aeronautics and space activities during the preceding calendar year." In recent years, the reports have been prepared on a fiscal year basis, consistent with the budgetary period now used in programs of the Federal Government. This year's report covers activities that took place from October 1, 1998, through September 30, 1999.

  3. Aeronautics and Space Report of the President: Fiscal Year 2001 Activities

    NASA Technical Reports Server (NTRS)

    2001-01-01

    The National Aeronautics and Space Act of 1958 directed the annual Aeronautics and Space Report to include a 'comprehensive description of the programmed activities and the accomplishments of all agencies of the United States in the field of aeronautics and space activities during the preceding calendar year.' In recent years the reports have been prepared on a fiscal-year basis consistent with the budgetary period now used in programs of the Federal Government. This year's report covers activities that took place from October 1, 2000, through September 30, 2001.

  4. Aeronautics and Space Report of the President: Fiscal Year 2000 Activities

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The National Aeronautics and Space Act of 1958 directed the annual Aeronautics and Space Report to include a "comprehensive description of the programmed activities and the accomplishments of all agencies of the United States in the field of aeronautics and space activities during the preceding calendar year." In recent years, the reports have been prepared on a fiscal year basis, consistent with the budgetary period now used in programs of the Federal Government. This year's report covers activities that took place from October 1, 1999, through September 30, 2000.

  5. Aeronautics and Space Report of the President: Fiscal Year 2005 Activities

    NASA Technical Reports Server (NTRS)

    2007-01-01

    The National Aeronautics and Space Act of 1958 directed the annual Aeronautics and Space Report to include a "comprehensive description of the programmed activities and the accomplishments of all agencies of the United States in the field of aeronautics and space activities during the preceding calendar year." In recent years, the reports have been prepared on a fiscal-year basis, consistent with the budgetary period now used in programs of the Federal Government. This year's report covers activities that took place from October 1 , 2004, through September 30, 2005.

  6. Aeronautics and Space Report of the President: Fiscal Year 2003 Activities

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The National Aeronautics and Space Act of 1958 directed the annual Aeronautics and Space Report to include a comprehensive description of the programmed activities and the accomplishments of all agencies of the United States in the field of aeronautics and space activities during the preceding calendar year. In recent years, the reports have been prepared on a fiscal-year basis, consistent with the budgetary period now used in programs of the Federal Government. This year's report covers activities that took place from October 1, 2002, through September 30, 2003.

  7. Aeronautics and Space Report of the President - Fiscal Year 2010 Activities

    NASA Technical Reports Server (NTRS)

    2011-01-01

    The National Aeronautics and Space Act of 1958 directed the annual Aeronautics and Space Report to include a "comprehensive description of the programmed activities and the accomplishments of all agencies of the United States in the field of aeronautics and space activities during the preceding calendar year." In recent years, the reports have been prepared on a fiscal-year basis, consistent with the budgetary period now used in programs of the Federal Government. This year's report covers activities that took place from October 1, 2009, through September 30, 2010.

  8. Mobile phone usage in complex urban systems: a space-time, aggregated human activity study

    NASA Astrophysics Data System (ADS)

    Tranos, Emmanouil; Nijkamp, Peter

    2015-04-01

    The present study aims to demonstrate the importance of digital data for investigating space-time dynamics of aggregated human activity in urban systems. Such dynamics can be monitored and modelled using data from mobile phone operators regarding mobile telephone usage. Using such an extensive dataset from the city of Amsterdam, this paper introduces space-time explanatory models of aggregated human activity patterns. Various modelling experiments and results are presented, which demonstrate that mobile telephone data are a good proxy of the space-time dynamics of aggregated human activity in the city.

  9. Aeronautics and Space Report of the President - Fiscal Year 2008 Activities

    NASA Technical Reports Server (NTRS)

    2009-01-01

    The National Aeronautics and Space Act of 1958 directed the annual Aeronautics and Space Report to include a "comprehensive description of the programmed activities and the accomplishments of all agencies of the United States in the field of aeronautics and space activities during the preceding calendar year." In recent years, the reports have been prepared on a fiscal-year basis, consistent with the budgetary period now used in programs of the Federal Government. This year's report covers activities that took place from October 1, 2007, through September 30, 2008.

  10. NASDA activities in space solar power system research, development and applications

    NASA Technical Reports Server (NTRS)

    Matsuda, Sumio; Yamamoto, Yasunari; Uesugi, Masato

    1993-01-01

    NASDA activities in solar cell research, development, and applications are described. First, current technologies for space solar cells such as Si, GaAs, and InP are reviewed. Second, future space solar cell technologies intended to be used on satellites of 21st century are discussed. Next, the flight data of solar cell monitor on ETS-V is shown. Finally, establishing the universal space solar cell calibration system is proposed.

  11. Virtual Reality: Developing a VR space for Academic activities

    NASA Astrophysics Data System (ADS)

    Kaimaris, D.; Stylianidis, E.; Karanikolas, N.

    2014-05-01

    Virtual reality (VR) is extensively used in various applications; in industry, in academia, in business, and is becoming more and more affordable for end users from the financial point of view. At the same time, in academia and higher education more and more applications are developed, like in medicine, engineering, etc. and students are inquiring to be well-prepared for their professional life after their educational life cycle. Moreover, VR is providing the benefits having the possibility to improve skills but also to understand space as well. This paper presents the methodology used during a course, namely "Geoinformatics applications" at the School of Spatial Planning and Development (Eng.), Aristotle University of Thessaloniki, to create a virtual School space. The course design focuses on the methods and techniques to be used in order to develop the virtual environment. In addition the project aspires to become more and more effective for the students and provide a real virtual environment with useful information not only for the students but also for any citizen interested in the academic life at the School.

  12. Wireless Video System for Extra Vehicular Activity in the International Space Station and Space Shuttle Orbiter Environment

    NASA Technical Reports Server (NTRS)

    Loh, Yin C.; Boster, John; Hwu, Shian; Watson, John C.; deSilva, Kanishka; Piatek, Irene (Technical Monitor)

    1999-01-01

    The Wireless Video System (WVS) provides real-time video coverage of astronaut extra vehicular activities during International Space Station (ISS) assembly. The ISS wireless environment is unique due to the nature of the ISS structure and multiple RF interference sources. This paper describes how the system was developed to combat multipath, blockage, and interference using an automatic antenna switching system. Critical to system performance is the selection of receiver antenna installation locations determined using Uniform Geometrical Theory of Diffraction (GTD) techniques.

  13. DNA replication origin activation in space and time.

    PubMed

    Fragkos, Michalis; Ganier, Olivier; Coulombe, Philippe; Méchali, Marcel

    2015-06-01

    DNA replication begins with the assembly of pre-replication complexes (pre-RCs) at thousands of DNA replication origins during the G1 phase of the cell cycle. At the G1-S-phase transition, pre-RCs are converted into pre-initiation complexes, in which the replicative helicase is activated, leading to DNA unwinding and initiation of DNA synthesis. However, only a subset of origins are activated during any S phase. Recent insights into the mechanisms underlying this choice reveal how flexibility in origin usage and temporal activation are linked to chromosome structure and organization, cell growth and differentiation, and replication stress.

  14. On-orbit Metrology and Calibration Requirements for Space Station Activities Definition Study

    NASA Technical Reports Server (NTRS)

    Cotty, G. M.; Ranganathan, B. N.; Sorrell, A. L.

    1989-01-01

    The Space Station is the focal point for the commercial development of space. The long term routine operation of the Space Station and the conduct of future commercial activities suggests the need for in-space metrology capabilities analogous when possible to those on-Earth. The ability to perform periodic calibrations and measurements with proper traceability is imperative for the routine operation of the Space Station. An initial review, however, indicated a paucity of data related to metrology and calibration requirements for in-space operations. This condition probably exists because of the highly developmental aspect of space activities to date, their short duration, and nonroutine nature. The on-orbit metrology and calibration needs of the Space Station were examined and assessed. In order to achieve this goal, the following tasks were performed: an up-to-date literature review; identification of on-orbit calibration techniques; identification of sensor calibration requirements; identification of calibration equipment requirements; definition of traceability requirements; preparation of technology development plans; and preparation of the final report. Significant information and major highlights pertaining to each task is presented. In addition, some general (generic) conclusions/observations and recommendations that are pertinent to the overall in-space metrology and calibration activities are presented.

  15. Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

    PubMed

    Heler, Robert; Samai, Poulami; Modell, Joshua W; Weiner, Catherine; Goldberg, Gregory W; Bikard, David; Marraffini, Luciano A

    2015-03-12

    Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

  16. Individualized Instruction in Science, Time-Space-Matter, Learning Activity Packages.

    ERIC Educational Resources Information Center

    Kuczma, R. M.

    Learning Activity Packages (LAP) relating to time, space, and matter are presented for use in sampling a new type of learning for a whole year. Besides the unit on introduction to individualized learning, 11 major topics are incorporated into three other units: (1) observation of the physical world, (2) space and exploration for environmental…

  17. Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage

    PubMed Central

    Josephs, Eric A.; Kocak, D. Dewran; Fitzgibbon, Christopher J.; McMenemy, Joshua; Gersbach, Charles A.; Marszalek, Piotr E.

    2015-01-01

    CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9's ability to be directed by single ‘guide RNA’ molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9's off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a ‘conformational gating’ mechanism driven by the interactions between the guide RNA and the 14th–17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex. PMID:26384421

  18. 14 CFR 1266.102 - Cross-waiver of liability for agreements for activities related to the International Space Station.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... for activities related to the International Space Station. 1266.102 Section 1266.102 Aeronautics and... liability for agreements for activities related to the International Space Station. (a) The objective of... exploration, exploitation, and use of outer space through the International Space Station (ISS). The...

  19. 14 CFR 1266.102 - Cross-waiver of liability for agreements for activities related to the International Space Station.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... for activities related to the International Space Station. 1266.102 Section 1266.102 Aeronautics and... liability for agreements for activities related to the International Space Station. (a) The objective of... exploration, exploitation, and use of outer space through the International Space Station (ISS). The...

  20. NASA. Marshall Space Flight Center Hydrostatic Bearing Activities

    NASA Technical Reports Server (NTRS)

    Benjamin, Theodore G.

    1991-01-01

    The basic approach for analyzing hydrostatic bearing flows at the Marshall Space Flight Center (MSFC) is briefly discussed. The Hydrostatic Bearing Team has responsibility for assessing and evaluating flow codes; evaluating friction, ignition, and galling effects; evaluating wear; and performing tests. The Office of Aerospace and Exploration Technology Turbomachinery Seals Tasks consist of tests and analysis. The MSFC in-house analyses utilize one-dimensional bulk-flow codes. Computational fluid dynamics (CFD) analysis is used to enhance understanding of bearing flow physics or to perform parametric analysis that are outside the bulk flow database. As long as the bulk flow codes are accurate enough for most needs, they will be utilized accordingly and will be supported by CFD analysis on an as-needed basis.

  1. Advanced Embedded Active Assemblies for Extreme Space Applications

    NASA Technical Reports Server (NTRS)

    DelCastillo, Linda; Moussessian, Alina; Mojarradi, Mohammad; Kolawa, Elizabeth

    2009-01-01

    This work describes the development and evaluation of advanced technologies for the integration of electronic die within membrane polymers. Specifically, investigators thinned silicon die, electrically connecting them with circuits on flexible liquid crystal polymer (LCP), using gold thermo-compression flip chip bonding, and embedding them within the material. Daisy chain LCP assemblies were thermal cycled from -135 to +85degC (Mars surface conditions for motor control electronics). The LCP assembly method was further utilized to embed an operational amplifier designed for operation within the Mars surface ambient. The embedded op-amp assembly was evaluated with respect to the influence of temperature on the operational characteristics of the device. Applications for this technology range from multifunctional, large area, flexible membrane structures to small-scale, flexible circuits that can be fit into tight spaces for flex to fit applications.

  2. 78 FR 40665 - Cost Accounting Standards: CAS 413 Pension Adjustments for Extraordinary Events

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-08

    ... Adjustments for Extraordinary Events AGENCY: Cost Accounting Standards Board, Office of Federal Procurement... Staff Discussion Paper (SDP) on CAS 413 Pension Adjustments for Extraordinary Events. This is the first... extraordinary events'' in the subject line. To ensure seating due to space constraints, potential attendees...

  3. Young adolescents' perceived activity space risk, peer networks, and substance use.

    PubMed

    Mason, Michael; Mennis, Jeremy; Way, Thomas; Light, John; Rusby, Julie; Westling, Erika; Crewe, Stephanie; Flay, Brian; Campbell, Leah; Zaharakis, Nikola; McHenry, Chantal

    2015-07-01

    Adolescent substance use is a developmentally contingent social practice that is constituted within the routine social-environment of adolescents' lives. Few studies have examined peer networks, perceived activity space risk (risk of substance use at routine locations), and substance use. We examined the moderating influence of peer network characteristics on the relationship between perceived activity space risk and substance use among a sample of 250 urban adolescents. Significant interactions were found between peer networks and perceived activity space risk on tobacco and marijuana use, such that protective peer networks reduced the effect of activity place risk on substance use. A significant 3-way interaction was found on marijuana use indicating that gender moderated peer network's effect on activity space risk. Conditional effect analysis found that boys' peer networks moderated the effect of perceived activity space risk on marijuana use, whereas for girls, the effect of perceived activity space risk on marijuana use was not moderated by their peer networks. These findings could advance theoretical models to inform social-environmental research among adolescents.

  4. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems

    PubMed Central

    Fonfara, Ines; Le Rhun, Anaïs; Chylinski, Krzysztof; Makarova, Kira S.; Lécrivain, Anne-Laure; Bzdrenga, Janek; Koonin, Eugene V.; Charpentier, Emmanuelle

    2014-01-01

    The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool. PMID:24270795

  5. Regulation of the Type I-F CRISPR-Cas system by CRP-cAMP and GalM controls spacer acquisition and interference.

    PubMed

    Patterson, Adrian G; Chang, James T; Taylor, Corinda; Fineran, Peter C

    2015-07-13

    The CRISPR-Cas prokaryotic 'adaptive immune systems' represent a sophisticated defence strategy providing bacteria and archaea with protection from invading genetic elements, such as bacteriophages or plasmids. Despite intensive research into their mechanism and application, how CRISPR-Cas systems are regulated is less clear, and nothing is known about the regulation of Type I-F systems. We used Pectobacterium atrosepticum, a Gram-negative phytopathogen, to study CRISPR-Cas regulation, since it contains a single Type I-F system. The CRP-cAMP complex activated the cas operon, increasing the expression of the adaptation genes cas1 and cas2-3 in addition to the genes encoding the Csy surveillance complex. Mutation of crp or cyaA (encoding adenylate cyclase) resulted in reductions in both primed spacer acquisition and interference. Furthermore, we identified a galactose mutarotase, GalM, which reduced cas operon expression in a CRP- and CyaA-dependent manner. We propose that the Type I-F system senses metabolic changes, such as sugar availability, and regulates cas genes to initiate an appropriate defence response. Indeed, elevated glucose levels reduced cas expression in a CRP- and CyaA-dependent manner. Taken together, these findings highlight that a metabolite-sensing regulatory pathway controls expression of the Type I-F CRISPR-Cas system to modulate levels of adaptation and interference.

  6. INCORPORATING ROUTINE ACTIVITIES, ACTIVITY SPACES, AND SITUATIONAL DEFINITIONS INTO THE SOCIAL SCHEMATIC THEORY OF CRIME*

    PubMed Central

    BARR, ASHLEY B.; LEI, MAN-KIT; STEWART, ERIC

    2014-01-01

    Simons and Burt’s (2011) social schematic theory (SST) of crime posits that adverse social factors are associated with offending because they promote a set of social schemas (i.e., a criminogenic knowledge structure) that elevates the probability of situational definitions favorable to crime. This study extends the SST model by incorporating the role of contexts for action. Furthermore, the study advances tests of the SST by incorporating a measure of criminogenic situational definitions to assess whether such definitions mediate the effects of schemas and contexts on crime. Structural equation models using 10 years of panel data from 582 African American youth provided strong support for the expanded theory. The results suggest that childhood and adolescent social adversity fosters a criminogenic knowledge structure as well as selection into criminogenic activity spaces and risky activities, all of which increase the likelihood of offending largely through situational definitions. Additionally, evidence shows that the criminogenic knowledge structure interacts with settings to amplify the likelihood of situational definitions favorable to crime. PMID:26392633

  7. Research on elastic large space structures as "plants' for active control

    NASA Technical Reports Server (NTRS)

    Ashley, H.; Vonflotow, A.

    1983-01-01

    Research on active control of large space structures is discussed. Intrinsic damping in monolithic metallic structures is discussed. Thermal relaxation and grain boundary relaxation are discussed, as are properties of thermal damping.

  8. The Living Ocean. SeaWiFS: Studying Ocean Color from Space. Teacher's Guide with Activities

    NASA Technical Reports Server (NTRS)

    1994-01-01

    This educational document, designed for grades 9 to 10, discusses the observation of oceans from space. Topics covered include ocean color, the role of phytoplankton, the carbon cycle, and the greenhouse effect. Activities and discussion questions are presented.

  9. Efficient targeted mutagenesis in soybean by TALENs and CRISPR/Cas9.

    PubMed

    Du, Hongyang; Zeng, Xuanrui; Zhao, Meng; Cui, Xiaopei; Wang, Qing; Yang, Hui; Cheng, Hao; Yu, Deyue

    2016-01-10

    Gene targeting (GT) is of great significance for advancing basic plant research and crop improvement. Both TALENs (transcription activator-like effectors nucleases) and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) systems have been developed for genome editing in eukaryotes, including crop plants. In this work, we present the comparative analysis of these two technologies for two soybean genome editing targets, GmPDS11 and GmPDS18. We found GT in soybean hairy roots with a single targeting efficiency range of 17.5-21.1% by TALENs, 11.7-18.1% by CRISPR/Cas9 using the AtU6-26 promoter, and 43.4-48.1% by CRISPR/Cas9 using the GmU6-16g-1 promoter, suggesting that the CRISPR/Cas9 using the GmU6-16g-1 promoter is probably a much more efficient tool compared to the other technologies. Similarly, our double mutation GT efficiency experiment with these three technologies displayed a targeting efficiency of 6.25% by TALENs, 12.5% by CRISPR/Cas9 using the AtU6-26 promoter, and 43.4-48.1% by CRISPR/Cas9 using the GmU6-16g-1 promoter, suggesting that CRISPR/Cas9 is still a better choice for simultaneous editing of multiple homoeoalleles. Furthermore, we observed albino and dwarf buds (PDS knock-out) by soybean transformation in cotyledon nodes. Our results demonstrated that both TALENs and CRISPR/Cas9 systems are powerful tools for soybean genome editing.

  10. Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9.

    PubMed

    Ceasar, S Antony; Rajan, Vinothkumar; Prykhozhij, Sergey V; Berman, Jason N; Ignacimuthu, S

    2016-09-01

    The clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR associated protein 9 (Cas9) system discovered as an adaptive immunity mechanism in prokaryotes has emerged as the most popular tool for the precise alterations of the genomes of diverse species. CRISPR/Cas9 system has taken the world of genome editing by storm in recent years. Its popularity as a tool for altering genomes is due to the ability of Cas9 protein to cause double-stranded breaks in DNA after binding with short guide RNA molecules, which can be produced with dramatically less effort and expense than required for production of transcription-activator like effector nucleases (TALEN) and zinc-finger nucleases (ZFN). This system has been exploited in many species from prokaryotes to higher animals including human cells as evidenced by the literature showing increasing sophistication and ease of CRISPR/Cas9 as well as increasing species variety where it is applicable. This technology is poised to solve several complex molecular biology problems faced in life science research including cancer research. In this review, we highlight the recent advancements in CRISPR/Cas9 system in editing genomes of prokaryotes, fungi, plants and animals and provide details on software tools available for convenient design of CRISPR/Cas9 targeting plasmids. We also discuss the future prospects of this advanced molecular technology.

  11. Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein.

    PubMed

    Jung, Chris J; Zhang, Junli; Trenchard, Elizabeth; Lloyd, Kent C; West, David B; Rosen, Barry; de Jong, Pieter J

    2017-04-01

    The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9-FokI. We then applied the optimized conditions to zygotes, delivering Cas9 as either mRNA or protein. We found that Cas9 nucleo-protein complex promotes highly efficient, multiplexed targeting of circular constructs containing reporter genes and floxed exons. This approach allows for a one-step zygote injection procedure targeting multiple genes to generate conditional alleles via homologous recombination, and simultaneous knockout of corresponding genes in non-targeted alleles via non-homologous end joining.

  12. Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration

    PubMed Central

    Kim, Kyoungmi; Park, Sung Wook; Kim, Jin Hyoung; Lee, Seung Hwan; Kim, Daesik; Koo, Taeyoung; Kim, Kwang-eun; Kim, Jeong Hun; Kim, Jin-Soo

    2017-01-01

    RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in nongenetic diseases is largely unexplored. Here, we focus on age-related macular degeneration (AMD), a leading cause of blindness in adults, which is associated with retinal overexpression of, rather than mutations in, the VEGFA gene. Subretinal injection of preassembled, Vegfa gene–specific Cas9 ribonucleoproteins (RNPs) into the adult mouse eye gave rise to mutagenesis at the target site in the retinal pigment epithelium. Furthermore, Cas9 RNPs effectively reduced the area of laser-induced choroidal neovascularization (CNV) in a mouse model of AMD. Genome-wide profiling of Cas9 off-target effects via Digenome-seq showed that off-target mutations were rarely induced in the human genome. Because Cas9 RNPs can function immediately after in vivo delivery and are rapidly degraded by endogenous proteases, their activities are unlikely to be hampered by antibody- and cell-mediated adaptive immune systems. Our results demonstrate that in vivo genome editing with Cas9 RNPs has the potential for the local treatment for nongenetic degenerative diseases, expanding the scope of RNA-guided genome surgery to a new dimension. PMID:28209587

  13. Vinculin-p130Cas interaction is critical for focal adhesion dynamics and mechano-transduction.

    PubMed

    Goldmann, Wolfgang H

    2014-03-01

    Adherent cells, when mechanically stressed, show a wide range of responses including large-scale changes in their mechanical behaviour and gene expression pattern. This is in part facilitated by activating the focal adhesion (FA) protein p130Cas through force-induced conformational changes that lead to the phosphorylation by src family kinases. Janostiak et al. [Janostiak et al. Cell Mol Life Sci (2013) DOI 10.1007/s00018-013-1450-x] have reported that the phosphorylation site Y12 on the SH3 domain of p130Cas modulates the binding with vinculin, a prominent mechano-coupling protein in FAs. Tension changes in FAs (due to the anchorage of the SH3 domain and C-terminal) bring about an extension of the substrate domain of p130Cas by unmasking the phosphorylation sites. These observations demonstrate that vinculin is an important modulator of the p130Cas-mediated mechano-transduction pathway in cells. The central aim should be now to test that vinculin is critical for p130Cas incorporation into the focal adhesion complex and for transmitting forces to the p130Cas molecule.

  14. Exploring the potential of genome editing CRISPR-Cas9 technology.

    PubMed

    Singh, Vijai; Braddick, Darren; Dhar, Pawan Kumar

    2017-01-30

    CRISPR-Cas9 is an RNA-mediated adaptive immune system that protects bacteria and archaea from viruses or plasmids. Herein we discuss the recent development of CRISPR-Cas9 into a key technology for genome editing, targeting, and regulation in a wide range of organisms and cell types. It requires a custom designed single guide-RNA (sgRNA), a Cas9 endonuclease, and PAM sequences in the target region. The sgRNA-Cas9 complex binds to its target and creates a double-strand break (DSB) that can be repaired by non-homologous end joining (NHEJ) or by the homology-directed repair (HDR) pathway, modifying or permanently replacing the genomic target sequence. Additionally, we highlight recent advances in the repurposing of CRISPR-Cas9 for repression, activation, and loci imaging. In this review, we underline the current progress and the future potential of the CRISPR-Cas9 system towards biomedical, therapeutic, industrial, and biotechnological applications.

  15. CRISPR-Cas9 for medical genetic screens: applications and future perspectives.

    PubMed

    Xue, Hui-Ying; Ji, Li-Juan; Gao, Ai-Mei; Liu, Ping; He, Jing-Dong; Lu, Xiao-Jie

    2016-02-01

    CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9) systems have emerged as versatile and convenient (epi)genome editing tools and have become an important player in medical genetic research. CRISPR-Cas9 and its variants such as catalytically inactivated Cas9 (dead Cas9, dCas9) and scaffold-incorporating single guide sgRNA (scRNA) have been applied in various genomic screen studies. CRISPR screens enable high-throughput interrogation of gene functions in health and diseases. Compared with conventional RNAi screens, CRISPR screens incur less off-target effects and are more versatile in that they can be used in multiple formats such as knockout, knockdown and activation screens, and can target coding and non-coding regions throughout the genome. This powerful screen platform holds the potential of revolutionising functional genomic studies in the near future. Herein, we introduce the mechanisms of (epi)genome editing mediated by CRISPR-Cas9 and its variants, introduce the procedures and applications of CRISPR screen in functional genomics, compare it with conventional screen tools and at last discuss current challenges and opportunities and propose future directions.

  16. CRISPR/Cas9-mediated genome engineering and the promise of designer flies on demand.

    PubMed

    Gratz, Scott J; Wildonger, Jill; Harrison, Melissa M; O'Connor-Giles, Kate M

    2013-01-01

    The CRISPR/Cas9 system has attracted significant attention for its potential to transform genome engineering. We and others have recently shown that the RNA-guided Cas9 nuclease can be employed to engineer the Drosophila genome, and that these modifications are efficiently transmitted through the germline. A single targeting RNA can guide Cas9 to a specific genomic sequence where it induces double-strand breaks that, when imperfectly repaired, yield mutations. We have also demonstrated that 2 targeting RNAs can be used to generate large defined deletions and that Cas9 can catalyze gene replacement by homologous recombination. Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have shown similar promise in Drosophila. However, the ease of producing targeting RNAs over the generation of unique sequence-directed nucleases to guide site-specific modifications makes the CRISPR/Cas9 system an appealingly accessible method for genome editing. From the initial planning stages, engineered flies can be obtained within a month. Here we highlight the variety of genome modifications facilitated by the CRISPR/Cas9 system along with key considerations for starting your own CRISPR genome engineering project.

  17. Thermal Technology Development Activities at the Goddard Space Flight Center - 2001

    NASA Technical Reports Server (NTRS)

    Butler, Dan

    2002-01-01

    This presentation provides an overview of thermal technology development activities carried out at NASA's Goddard Space Flight Center during 2001. Specific topics covered include: two-phase systems (heat pipes, capillary pumped loops, vapor compression systems and phase change materials), variable emittance systems, advanced coatings, high conductivity materials and electrohydrodynamic (EHD) thermal coatings. The application of these activities to specific space missions is also discussed.

  18. Active Control of Flexible Space Structures Using the Nitinol Shape Memory Actuators

    DTIC Science & Technology

    1987-10-01

    number) FIELD !GROUP SUBGROUP I Active Control, Nitinol Actuators, Space Structures 9. ABSTRACT (Continue on reverse if necessary and identify by block...number) Summarizes research progress in the feasibility demonstration of active vibration control using Nitinol shape memory actuators. Tests on...FLEXIBLE SPACE STRUCTURES USING NITINOL SHAPE MEMORY ACTUATORS FINAL REPORT FOR PHASE I SDIO CONTRACT #F49620-87-C-0035 0 BY DR. AMR M. BAZ KARIM R

  19. Assessment of MSFCs Process for the Development and Activation of Space Act Agreement

    NASA Technical Reports Server (NTRS)

    Daugherty, Rachel

    2014-01-01

    Space Act Agreements (SAAs) are contractual agreements that NASA utilizes to form partnerships with researchers, industry, and academia to stimulate cutting-edge innovation within the science and technology communities. center dot This study assessed the current SAA development and activation process at Marshall Space Flight Center (MSFC) to determine if improvements could be implemented to increase productivity, decrease time to activation, and improve the quality of deliverables.

  20. Sky Luminaries in the Space Orienting Activity of Homo Sapiens in the Middle Palaeolithic

    NASA Astrophysics Data System (ADS)

    Kaurov, E. N.

    Data describing the beginnings of the space orienting activity of Homo sapiens is analysed and systematized: observation of the Pole and the recognition of Ursa Major were used as the basis of the determination of the points of the compass. Data and results from astronomy, history of astronomy, archaeology and palaeoanthropology were used for the reconstruction of the evolution of the space orienting activity of Homo sapiens.

  1. CRISPR-Cas: biology, mechanisms and relevance

    PubMed Central

    Hille, Frank

    2016-01-01

    Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes—termed spacers—into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical processes behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR-Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent. This article is part of the themed issue ‘The new bacteriology’. PMID:27672148

  2. Life Extension Activities for the Hubble Space Telescope

    NASA Technical Reports Server (NTRS)

    Walyus, Keith D.; Pepe, Joyce A. K.; Prior, Michael

    2004-01-01

    With the cancellation of the Hubble Space Telescope (HST) Servicing Mission 4 (SM4), the HST Project will face numerous challenges to keep the Telescope operating during the remainder of the decade. As part of the SM4, the HST Project had planned to install various upgrades to the Telescope including the installation of new batteries and new rate integrating gyros. Without these upgrades, reliability analysis indicates that the spacecraft will lose the capability to conduct science operations later this decade. The HST team will be severely challenged to maximize the Telescope's remaining operational lifetime, while still trying to maximize - its science output and quality. Two of the biggest areas of concern are the age and condition of the batteries and gyros. Together they offer the largest potential extension in Telescope lifetime and present the biggest challenges to the HST team. The six Ni-H batteries on HST are the original batteries from launch. With fourteen years of operational life, these batteries have collectively lasted longer than any other comparable mission. Yet as with all batteries, their capacity has been declining. Engineers are examining various methods to prolong the life of these mission critical batteries, and retard the rate of degradation. This paper will focus on these and other efforts to prolong the life of the HST, thus enabling it to remain a world-class observatory for as long as possible.

  3. Visualization analysis of CRISPR/Cas9 gene editing technology studies*

    PubMed Central

    Du, Quan-sheng; Cui, Jie; Zhang, Chun-jie; He, Ke

    2016-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is an adaptive immune defense system that resists the invasion of viruses and plasmids heterologous genetic material in bacteria and archaea. Taking the literature related to gene editing technology of CRISPR/Cas9 from the Web of Science database from 2002 to 2015, we use the software CiteSpaceV to analyze co-cited literature in order to establish the research hotspots and fronts recently in this field by knowledge mapping. PMID:27704749

  4. Visualization analysis of CRISPR/Cas9 gene editing technology studies.

    PubMed

    Du, Quan-Sheng; Cui, Jie; Zhang, Chun-Jie; He, Ke

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is an adaptive immune defense system that resists the invasion of viruses and plasmids heterologous genetic material in bacteria and archaea. Taking the literature related to gene editing technology of CRISPR/Cas9 from the Web of Science database from 2002 to 2015, we use the software CiteSpaceV to analyze co-cited literature in order to establish the research hotspots and fronts recently in this field by knowledge mapping.

  5. Genome engineering using the CRISPR-Cas9 system.

    PubMed

    Ran, F Ann; Hsu, Patrick D; Wright, Jason; Agarwala, Vineeta; Scott, David A; Zhang, Feng

    2013-11-01

    Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.

  6. Structural Dynamics Experimental Activities in Ultra-Lightweight and Inflatable Space Structures

    NASA Technical Reports Server (NTRS)

    Pappa, Richard S.; Lassiter, John O.; Ross, Brian P.

    2001-01-01

    This paper reports recently completed structural dynamics experimental activities with new ultra-lightweight and inflatable space structures (a.k.a., "Gossamer" spacecraft) at NASA Langley Research Center, NASA Marshall Space Flight Center, and NASA Goddard Space Flight Center. Nine aspects of this work are covered: 1) inflated, rigidized tubes, 2) active control experiments, 3) photogrammetry, 4) laser vibrometry, 5) modal tests of inflatable structures, 6) in-vacuum modal tests, 7) tensioned membranes, 8) deployment tests, and 9) flight experiment support. Structural dynamics will play a major role in the design and eventually in-space deployment and performance of Gossamer spacecraft. Experimental research and development such as this is required to validate new analysis methods. The activities discussed in the paper are pathfinder accomplishments. conducted on unique components and prototypes of future spacecraft systems.

  7. Structural Dynamics Experimental Activities in Ultra-Lightweight and Inflatable Space Structures

    NASA Technical Reports Server (NTRS)

    Pappa, Richard S.; Lassiter, John O.; Ross, Brian P.

    2001-01-01

    This paper reports recently completed structural dynamics experimental activities with new ultralightweight and inflatable space structures (a.k.a., "Gossamer" spacecraft) at NASA Langley Research Center, NASA Marshall Space Flight Center, and NASA Goddard Space Flight Center. Nine aspects of this work are covered, as follows: 1) inflated, rigidized tubes, 2) active control experiments, 3) photogrammetry, 4) laser vibrometry, 5) modal tests of inflatable structures, 6) in-vacuum modal tests, 7) tensioned membranes, 8) deployment tests, and 9) flight experiment support. Structural dynamics will play a major role in the design and eventual in-space deployment and performance of Gossamer spacecraft, and experimental R&D work such as this is required now to validate new analytical prediction methods. The activities discussed in the paper are pathfinder accomplishments, conducted on unique components and prototypes of future spacecraft systems.

  8. Enzyme activities and membrane lipids in artemia cysts after a long duration space flight

    NASA Astrophysics Data System (ADS)

    Gaubin, Y.; Prévost, M. C.; Cariven, C.; Pianezzi, B.; Planel, H.; Soleilhavoup, J. P.

    1996-01-01

    In the Free Flyer Biostack Experiment (L.D.E.F. mission) investigations have shown that biological objects in a resting state can survive more than 5.5 years of exposure to the space factors in particular microgravity and cosmic rays. We have measured enzyme activities involved in metabolic pathways of sugar and lipid degradation and determined phospholipid composition. Pyruvate kinase and glucose-6-phosphate dehydrogenase activities in space-exposed cysts were higher than in earth controls after 1 hour incubation. In controls, total phospholipids remained unchanged, on the contrary they increased significantly in space-exposed cysts. The rate of metabolism of various phospholipid components was unchanged in controls allowing the development while the level of most of them decreased in space-exposed cysts except for phosphatidylcholine. Enzyme activities (acetylhydrolase, phospholipase A_2 and lyso phospholipase) involved in phospholipid degradation increased ; however, activities were much higher in space-exposed cysts. In conclusion, the long duration space flight resulted in an increase of the metabolic activity correlated with a faster development within the first 20 hours of post flight incubation.

  9. Investigation of brightness changes of MZ Cas and TZ Cas in B- and V-light

    NASA Technical Reports Server (NTRS)

    Lukatskaya, F. I.; Kheylo, E. S.

    1973-01-01

    The results are presented concerning statistical processing of two-color observations of MZ Cas and TZ Cas. Light histograms, dispersion and statistical amplitudes are given. Light variations of the variables are represented by normal stochastic processes. Observational data are tabulated.

  10. Putting the CAS Standards to Work. Training Manual for the CAS Self Assessment Guides.

    ERIC Educational Resources Information Center

    Yerian, Jean M.; Miller, Theodore K., Ed.

    These 18 self-assessment guides and training manual from the Council for the Advancement of Standards (CAS) for Student Services/Development Programs translate the CAS Standards and Guidelines of 1986 into a format for self-study purposes. These self-study guides allow an institution to assure compliance with minimally-acceptable practice, gain an…

  11. CDPP activities: Promoting research and education in space physics

    NASA Astrophysics Data System (ADS)

    Genot, V. N.; Andre, N.; Cecconi, B.; Gangloff, M.; Bouchemit, M.; Dufourg, N.; Pitout, F.; Budnik, E.; Lavraud, B.; Rouillard, A. P.; Heulet, D.; Bellucci, A.; Durand, J.; Delmas, D.; Alexandrova, O.; Briand, C.; Biegun, A.

    2015-12-01

    The French Plasma Physics Data Centre (CDPP, http://cdpp.eu/) addresses for more than 15 years all issues pertaining to natural plasma data distribution and valorization. Initially established by CNES and CNRS on the ground of a solid data archive, CDPP activities diversified with the advent of broader networks and interoperability standards, and through fruitful collaborations (e.g. with NASA/PDS): providing access to remote data, designing and building science driven analysis tools then became at the forefront of CDPP developments. For instance today AMDA helps scientists all over the world accessing and analyzing data from ancient to very recent missions (from Voyager, Galileo, Geotail, ... to Maven, Rosetta, MMS, ...) as well as results from models and numerical simulations. Other tools like the Propagation Tool or 3DView allow users to put their data in context and interconnect with other databases (CDAWeb, MEDOC) and tools (Topcat). This presentation will briefly review this evolution, show technical and science use cases, and finally put CDPP activities in the perspective of ongoing collaborative projects (Europlanet H2020, HELCATS, ...) and future missions (Bepicolombo, Solar Orbiter, ...).

  12. Lunar bases and space activities of the 21st century

    NASA Technical Reports Server (NTRS)

    Mendell, W. W. (Editor)

    1985-01-01

    The present conference gives attention to such major aspects of lunar colonization as lunar base concepts, lunar transportation, lunar science research activities, moon-based astronomical researches, lunar architectural construction, lunar materials and processes, lunar oxygen production, life support and health maintenance in lunar bases, societal aspects of lunar colonization, and the prospects for Mars colonization. Specific discussions are presented concerning the role of nuclear energy in lunar development, achromatic trajectories and the industrial scale transport of lunar resources, advanced geologic exploration from a lunar base, geophysical investigations of the moon, moon-based astronomical interferometry, the irradiation of the moon by particles, cement-based composites for lunar base construction, electrostatic concentration of lunar soil minerals, microwave processing of lunar materials, a parametric analysis of lunar oxygen production, hydrogen from lunar regolith fines, metabolic support for a lunar base, past and future Soviet lunar exploration, and the use of the moons of Mars as sources of water for lunar bases.

  13. Space-Based Detection of Sinkhole Activity in Central Florida

    NASA Astrophysics Data System (ADS)

    Oliver-Cabrera, T.; Kruse, S.; Wdowinski, S.

    2015-12-01

    Central Florida's thick carbonate deposits and hydrological conditions have made the area prone to sinkhole development. Sinkhole collapse is a major geologic hazard in central Florida threatening human life and causing substantial damage to property. According to the Florida Senate report in 2010, between 2006-2010 total insurance claims due to sinkhole activity were around $200 million per year. Detecting sinkhole deformation before a collapse is a very difficult task, due to small or sometimes unnoticeable surface changes. Most techniques used to monitor sinkholes provide very localized information and cannot be implemented to study broad areas. This is the case of central Florida, where the active zone spans over hundreds of square-kilometers. In this study we use Interferometric Synthetic Aperture Radar (InSAR) observations acquired over several locations in central Florida to detect possible pre-collapse deformation. The study areas were selected because they have shown suspicious sinkhole behavior. One of the sites collapsed on March 2013 destroying a property and killing a man. To generate the InSAR results we use six datasets acquired by the TerraSAR-X and Cosmo-SkyMed satellites with various acquisition modes reflecting pixel resolutions between 25cm and 2m. Preliminary InSAR results show good coherence over constructed areas and low coherence in vegetated zones, justifying our analysis that focuses on the man-made structures. After full datasets will be acquired, a Persistent Scatterer Interferometry (PSI) time series analysis will be performed for detecting localized deformation at spatial scale of 1-5 meters. The project results will be verified using Ground Penetrating Radar.

  14. A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium.

    PubMed

    Palanisamy, Arun P; Suryakumar, Geetha; Panneerselvam, Kavin; Willey, Christopher D; Kuppuswamy, Dhandapani

    2015-12-01

    Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src's adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either β-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24-48 h PO myocardium. Our studies indicate that c-Src's adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium.

  15. I can see CRISPR now, even when phage are gone: a view on alternative CRISPR-Cas functions from the prokaryotic envelope

    PubMed Central

    Ratner, Hannah K.; Sampson, Timothy R.; Weiss, David S.

    2015-01-01

    Purpose CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids that adapt as new environmental threats arise. There are emerging examples of CRISPR-Cas functions in bacterial physiology beyond their role in adaptive immunity. This highlights the poorly understood, but potentially common, moonlighting functions of these abundant systems. We propose that these non-canonical CRISPR-Cas activities have evolved to respond to stresses at the cell envelope. Recent findings Here, we discuss recent literature describing the impact of the extracellular environment on the regulation of CRISPR-Cas systems, and the influence of CRISPR-Cas activity on bacterial physiology. The described non-canonical CRISPR-Cas functions allow the bacterial cell to respond to the extracellular environment, primarily through changes in envelope physiology. Summary This review discusses the expanding non-canonical functions of CRISPR-Cas systems, including their roles in virulence, focusing mainly on their relationship to the cell envelope. We first examine the effects of the extracellular environment on regulation of CRISPR-Cas components, and then discuss the impact of CRISPR-Cas systems on bacterial physiology, focusing on their roles in influencing interactions with the environment including host organisms. PMID:25887612

  16. Redefining Neighborhoods Using Common Destinations: Social Characteristics of Activity Spaces and Home Census Tracts Compared

    PubMed Central

    Jones, Malia; Pebley, Anne R.

    2014-01-01

    Research on neighborhood effects has focused largely on residential neighborhoods, but people are exposed to many other places in the course of their daily lives—at school, at work, when shopping, and so on. Thus, studies of residential neighborhoods consider only a subset of the social-spatial environment affecting individuals. In this article, we examine the characteristics of adults’ “activity spaces”—spaces defined by locations that individuals visit regularly, in Los Angeles County, California. Using geographic information system (GIS) methods, we define activity spaces in two ways and estimate their socioeconomic characteristics. Our research has two goals. First, we determine whether residential neighborhoods represent the social conditions to which adults are exposed in the course of their regular activities. Second, we evaluate whether particular groups are exposed to a broader or narrower range of social contexts in the course of their daily activities. We find that activity spaces are substantially more heterogeneous in terms of key social characteristics, compared to residential neighborhoods. However, the characteristics of both home neighborhoods and activity spaces are closely associated with individual characteristics. Our results suggest that most people experience substantial segregation across the range of spaces in their daily lives, not just at home. PMID:24719273

  17. Active space debris removal by a hybrid propulsion module

    NASA Astrophysics Data System (ADS)

    DeLuca, L. T.; Bernelli, F.; Maggi, F.; Tadini, P.; Pardini, C.; Anselmo, L.; Grassi, M.; Pavarin, D.; Francesconi, A.; Branz, F.; Chiesa, S.; Viola, N.; Bonnal, C.; Trushlyakov, V.; Belokonov, I.

    2013-10-01

    During the last 40 years, the mass of the artificial objects in orbit increased quite steadily at the rate of about 145 metric tons annually, leading to a total tally of approximately 7000 metric tons. Now, most of the cross-sectional area and mass (97% in LEO) is concentrated in about 4600 intact objects, i.e. abandoned spacecraft and rocket bodies, plus a further 1000 operational spacecraft. Simulations and parametric analyses have shown that the most efficient and effective way to prevent the outbreak of a long-term exponential growth of the catalogued debris population would be to remove enough cross-sectional area and mass from densely populated orbits. In practice, according to the most recent NASA results, the active yearly removal of approximately 0.1% of the abandoned intact objects would be sufficient to stabilize the catalogued debris in low Earth orbit, together with the worldwide adoption of mitigation measures. The candidate targets for removal would have typical masses between 500 and 1000 kg, in the case of spacecraft, and of more than 1000 kg, in the case of rocket upper stages. Current data suggest that optimal active debris removal missions should be carried out in a few critical altitude-inclination bands. This paper deals with the feasibility study of a mission in which the debris is removed by using a hybrid propulsion module as propulsion unit. Specifically, the engine is transferred from a servicing platform to the debris target by a robotic arm so to perform a controlled disposal. Hybrid rocket technology for de-orbiting applications is considered a valuable option due to high specific impulse, intrinsic safety, thrust throttle ability, low environmental impact and reduced operating costs. Typically, in hybrid rockets a gaseous or liquid oxidizer is injected into the combustion chamber along the axial direction to burn a solid fuel. However, the use of tangential injection on a solid grain Pancake Geometry allows for more compact design of

  18. Autonomous Sensorweb Operations for Integrated Space, In-Situ Monitoring of Volcanic Activity

    NASA Technical Reports Server (NTRS)

    Chien, Steve A.; Doubleday, Joshua; Kedar, Sharon; Davies, Ashley G.; Lahusen, Richard; Song, Wenzhan; Shirazi, Behrooz; Mandl, Daniel; Frye, Stuart

    2010-01-01

    We have deployed and demonstrated operations of an integrated space in-situ sensorweb for monitoring volcanic activity. This sensorweb includes a network of ground sensors deployed to the Mount Saint Helens volcano as well as the Earth Observing One spacecraft. The ground operations and space operations are interlinked in that ground-based intelligent event detections can cause the space segment to acquire additional data via observation requests and space-based data acquisitions (thermal imagery) can trigger reconfigurations of the ground network to allocate increased bandwidth to areas of the network best situated to observe the activity. The space-based operations are enabled by an automated mission planning and tasking capability which utilizes several Opengeospatial Consortium (OGC) Sensorweb Enablement (SWE) standards which enable acquiring data, alerts, and tasking using web services. The ground-based segment also supports similar protocols to enable seamless tasking and data delivery. The space-based segment also supports onboard development of data products (thermal summary images indicating areas of activity, quicklook context images, and thermal activity alerts). These onboard developed products have reduced data volume (compared to the complete images) which enables them to be transmitted to the ground more rapidly in engineering channels.

  19. Individualized Instruction in Science, Earth-Space Project, Self-Directed Activities.

    ERIC Educational Resources Information Center

    Kuczma, R. M.

    As a supplement to Learning Activity Packages (LAP) of the earth-space project, this manual presents self-directed activities especially designed for individualized instruction. Besides an introduction to LAP characteristics, sets of instructions are given in connection with the metric system, the earth's dimensions, indirect evidence for atomic…

  20. Centralising Space: The Physical Education and Physical Activity Experiences of South Asian, Muslim Girls

    ERIC Educational Resources Information Center

    Stride, Annette

    2016-01-01

    This paper explores the physical education (PE) and physical activity experiences of a group of South Asian, Muslim girls, a group typically marginalised in PE and physical activity research. The study responds to ongoing calls for research to explore across different spaces in young people's lives. Specifically, I draw on a…

  1. Games and Activities for Severely Handicapped Students Utilizing Small Space and Minimal Equipment.

    ERIC Educational Resources Information Center

    Aharoni, Hezkiah

    1982-01-01

    The manual describes games and activities requiring minimal equipment and space for severely handicapped children. General guidelines touch upon such aspects as the importance of avoiding frustration or boredom, varying the activity to maintain interest, providing short rest periods when necessary, and ensuring the fullest possible participation…

  2. Individualized Instruction in Science, Time-Space-Matter, Self-Directed Activities.

    ERIC Educational Resources Information Center

    Kuczma, R. M.

    As a supplement to Learning Activity Packages (LAP) on the time-space-matter subject, details are presented for self-directed activities. Major descriptions are given on the background of LAP characteristics, metric system, profile graph construction, spectroscope operation, radiant energy measurement, sunspot effects, density determination,…

  3. Communication: Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    SciTech Connect

    Parker, Shane M.; Shiozaki, Toru

    2014-12-07

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few μE{sub h} or less) with M = 128 in both cases. This rapid convergence is because the renormalization steps are used only for the interfragment electron correlation.

  4. Effects of Space Weather on Biomedical Parameters during the Solar Activity Cycles 23-24.

    PubMed

    Ragul'skaya, M V; Rudenchik, E A; Chibisov, S M; Gromozova, E N

    2015-06-01

    The results of long-term (1998-2012) biomedical monitoring of the biotropic effects of space weather are discussed. A drastic change in statistical distribution parameters in the middle of 2005 was revealed that did not conform to usual sinusoidal distribution of the biomedical data reflecting changes in the number of solar spots over a solar activity cycle. The dynamics of space weather of 2001-2012 is analyzed. The authors hypothesize that the actual change in statistical distributions corresponds to the adaptation reaction of the biosphere to nonstandard geophysical characteristics of the 24th solar activity cycle and the probable long-term decrease in solar activity up to 2067.

  5. Meeting the Grand Challenge of Protecting Astronauts Health: Electrostatic Active Space Radiation Shielding for Deep Space Missions

    NASA Technical Reports Server (NTRS)

    Tripathi, Ram K.

    2016-01-01

    This report describes the research completed during 2011 for the NASA Innovative Advanced Concepts (NIAC) project. The research is motivated by the desire to safely send humans in deep space missions and to keep radiation exposures within permitted limits. To this end current material shielding, developed for low earth orbit missions, is not a viable option due to payload and cost penalties. The active radiation shielding is the path forward for such missions. To achieve active space radiation shielding innovative large lightweight gossamer space structures are used. The goal is to deflect enough positive ions without attracting negatively charged plasma and to investigate if a charged Gossamer structure can perform charge deflections without significant structural instabilities occurring. In this study different innovative configurations are explored to design an optimum active shielding. In addition, to establish technological feasibility experiments are performed with up to 10kV of membrane charging, and an electron flux source with up to 5keV of energy and 5mA of current. While these charge flux energy levels are much less than those encountered in space, the fundamental coupled interaction of charged Gossamer structures with the ambient charge flux can be experimentally investigated. Of interest are, will the EIMS remain inflated during the charge deflections, and are there visible charge flux interactions. Aluminum coated Mylar membrane prototype structures are created to test their inflation capability using electrostatic charging. To simulate the charge flux, a 5keV electron emitter is utilized. The remaining charge flux at the end of the test chamber is measured with a Faraday cup mounted on a movable boom. A range of experiments with this electron emitter and detector were performed within a 30x60cm vacuum chamber with vacuum environment capability of 10-7 Torr. Experiments are performed with the charge flux aimed at the electrostatically inflated

  6. Overview of CRISPR-Cas9 Biology.

    PubMed

    Ratner, Hannah K; Sampson, Timothy R; Weiss, David S

    2016-12-01

    Prokaryotes use diverse strategies to improve fitness in the face of different environmental threats and stresses, including those posed by mobile genetic elements (e.g., bacteriophages and plasmids). To defend against these elements, many bacteria and archaea use elegant, RNA-directed, nucleic acid-targeting adaptive restriction machineries called CRISPR -: Cas (CRISPR-associated) systems. While providing an effective defense against foreign genetic elements, these systems have also been observed to play critical roles in regulating bacterial physiology during environmental stress. Increasingly, CRISPR-Cas systems, in particular the Type II systems containing the Cas9 endonuclease, have been exploited for their ability to bind desired nucleic acid sequences, as well as direct sequence-specific cleavage of their targets. Cas9-mediated genome engineering is transcending biological research as a versatile and portable platform for manipulating genetic content in myriad systems. Here, we present a systematic overview of CRISPR-Cas history and biology, highlighting the revolutionary tools derived from these systems, which greatly expand the molecular biologists' toolkit.

  7. Harpoon technology development for the active removal of space debris

    NASA Astrophysics Data System (ADS)

    Dudziak, Roger; Tuttle, Sean; Barraclough, Simon

    2015-08-01

    This paper presents the results of preliminary empirical testing and numerical modelling carried out to demonstrate the effectiveness of using a harpoon in an ADR application. Empirical testing involving the impact of blunt and conical shaped steel tips into 3 mm Al plate showed that the ballistic limit varies in proportion to the tip circumference, with conical shapes resulting in a higher relative ballistic limit due to the additional energy required for petaling. The creation of secondary debris was also monitored. It was found that blunt shapes created a plug during penetration as a result of shearing around the periphery of the projectile, whilst conical tips resulted in minor spalling and fragmentation. Preliminary oblique impact testing with conical and blunt tips showed that the ballistic limit increases with obliquity at a greater rate for blunt tips than conical ones. Impact testing of 3 mm Al plate with conical projectiles at low temperatures showed a more brittle fracture mode when compared with targets impacted at room temperature. As such, the fragmentation and spalling evident in room temperature targets was absent. The energy required to perforate the cooled plates also increased. Impact testing of Al panel obstructed with fixed heat pipes showed that the harpoon could successfully penetrate a target panel with such an obstruction due to shearing of the pipe flange. Testing of two lock on mechanisms showed that both a spring activated and integrated toggle could reliably open upon impact. This testing also used a tensile testing machine to show that both designs could withstand the force expected during deorbiting manoeuvres after impact with Al H/C panels. A parametric simulation comparing the diameter of conical tips with ballistic limits showed a good agreement with the predictions of De Marre's formula for normal impact. This suggests that the ballistic limit of plates impacted by conical projectiles can be successfully extrapolated with limited

  8. 26 CFR 1.863-8 - Source of income derived from space and ocean activity under section 863(d).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 9 2013-04-01 2013-04-01 false Source of income derived from space and ocean... to Taxable Years Prior to December 30, 1996 § 1.863-8 Source of income derived from space and ocean... space and ocean activity (space and ocean income) is sourced under the rules of this...

  9. 26 CFR 1.863-8 - Source of income derived from space and ocean activity under section 863(d).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 9 2012-04-01 2012-04-01 false Source of income derived from space and ocean... to Taxable Years Prior to December 30, 1996 § 1.863-8 Source of income derived from space and ocean... space and ocean activity (space and ocean income) is sourced under the rules of this...

  10. 26 CFR 1.863-8 - Source of income derived from space and ocean activity under section 863(d).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 9 2014-04-01 2014-04-01 false Source of income derived from space and ocean... to Taxable Years Prior to December 30, 1996 § 1.863-8 Source of income derived from space and ocean... space and ocean activity (space and ocean income) is sourced under the rules of this...

  11. 14 CFR 1266.104 - Cross-waiver of liability for launch agreements for science or space exploration activities...

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... agreements for science or space exploration activities unrelated to the International Space Station. 1266.104... LIABILITY § 1266.104 Cross-waiver of liability for launch agreements for science or space exploration... cross-waiver of liability between the parties to agreements for NASA's science or space...

  12. 26 CFR 1.863-8 - Source of income derived from space and ocean activity under section 863(d).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 9 2011-04-01 2011-04-01 false Source of income derived from space and ocean... to Taxable Years Prior to December 30, 1996 § 1.863-8 Source of income derived from space and ocean... space and ocean activity (space and ocean income) is sourced under the rules of this...

  13. 14 CFR 1266.104 - Cross-waiver of liability for launch agreements for science or space exploration activities...

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... agreements for science or space exploration activities unrelated to the International Space Station. 1266.104... LIABILITY § 1266.104 Cross-waiver of liability for launch agreements for science or space exploration... cross-waiver of liability between the parties to agreements for NASA's science or space...

  14. 14 CFR 1266.104 - Cross-waiver of liability for launch agreements for science or space exploration activities...

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... agreements for science or space exploration activities unrelated to the International Space Station. 1266.104... LIABILITY § 1266.104 Cross-waiver of liability for launch agreements for science or space exploration... cross-waiver of liability between the parties to agreements for NASA's science or space...

  15. 14 CFR 1266.104 - Cross-waiver of liability for launch agreements for science or space exploration activities...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... agreements for science or space exploration activities unrelated to the International Space Station. 1266.104... LIABILITY § 1266.104 Cross-waiver of liability for launch agreements for science or space exploration... cross-waiver of liability between the parties to agreements for NASA's science or space...

  16. The INAF contribution to the ASI Space Debris program: observational activities.

    NASA Astrophysics Data System (ADS)

    Pupillo, G.; Salerno, E.; Bartolini, M.; Di Martino, M.; Mattana, A.; Montebugnoli, S.; Portelli, C.; Pluchino, S.; Schillirò, F.; Konovalenko, A.; Nabatov, A.; Nechaeva, M.

    Space debris are man made objects orbiting around Earth that pose a serious hazard for both present and future human activities in space. Since 2007 the Istituto Nazionale di Astrofisica (INAF) carried out a number of radar campaigns in the framework of the ASI ``Space Debris'' program. The observations were performed by using bi- and multi-static radars, composed of the INAF 32-m Italian radiotelescopes located at Medicina and Noto (used as receivers) and the 70-m parabolic antenna at Evpatoria (Ukraine) used as transmitter. The 32 m Ventspils antenna in Latvia also participated in the last campaign at the end of June 2010. Several kinds of objects in various orbital regions (radar calibrators, rocket upper stages, debris of different sizes) were observed and successfully detected. Some unknown objects were also discovered in LEO during the beam-park sessions. In this paper we describe some results of the INAF-ASI space debris research activity.

  17. Breast Cancer Antiestrogen Resistance 3 (BCAR3) – p130Cas Interactions Promote Adhesion Disassembly and Invasion in Breast Cancer Cells

    PubMed Central

    Cross, Allison M.; Wilson, Ashley L.; Guerrero, Michael S.; Thomas, Keena S.; Bachir, Alexia I.; Kubow, Kristopher E.; Horwitz, A. Rick; Bouton, Amy H.

    2016-01-01

    Adhesion turnover is critical for cell motility and invasion. We previously demonstrated that the adaptor molecule Breast Cancer Antiestrogen Resistance 3 (BCAR3) promotes adhesion disassembly and breast tumor cell invasion. One of two established binding partners of BCAR3 is the adaptor molecule, p130Cas. In this study, we sought to determine whether signaling through the BCAR3/Cas complex was responsible for the cellular functions of BCAR3. We show that the entire pool of BCAR3 is in complex with Cas in invasive breast tumor cells and that these proteins co-localize in dynamic cellular adhesions. While accumulation of BCAR3 in adhesions did not require Cas binding, a direct interaction between BCAR3 and Cas was necessary for efficient dissociation of BCAR3 from adhesions. The dissociation rates of Cas and two other adhesion molecules, α-actinin and talin, were also significantly slower in the presence of a Cas-binding mutant of BCAR3, suggesting that turnover of the entire adhesion complex was delayed under these conditions. As was the case for adhesion turnover, BCAR3-Cas interactions were found to be important for BCAR3-mediated breast tumor cell chemotaxis toward serum and invasion in Matrigel. Previous work demonstrated that BCAR3 is a potent activator of Rac1, which in turn is an important regulator of adhesion dynamics and invasion. However, in contrast to wildtype BCAR3, ectopic expression of the Cas-binding mutant of BCAR3 failed to induce Rac1 activity in breast cancer cells. Together, these data show that the ability of BCAR3 to promote adhesion disassembly, tumor cell migration and invasion, and Rac1 activity is dependent on its ability to bind to Cas. The activity of BCAR3-Cas complexes as a functional unit in breast cancer is further supported by the co-expression of these molecules in multiple subtypes of human breast tumors. PMID:27109104

  18. Smart SPHERES: A Telerobotic Free-Flyer for Intravehicular Activities in Space

    NASA Technical Reports Server (NTRS)

    Fong, Terrence; Micire, Mark J.; Morse, Ted; Park, Eric; Provencher, Chris; To, Vinh; Wheeler, D. W.; Mittman, David; Torres, R. Jay; Smith, Ernest

    2013-01-01

    Smart SPHERES is a prototype free-flying space robot based on the SPHERES platform. Smart SPHERES can be remotely operated by astronauts inside a spacecraft, or by mission controllers on the ground. We developed Smart SPHERES to perform a variety of intravehicular activities (IVA), such as operations inside the International Space Station (ISS). These IVA tasks include environmental monitoring surveys (radiation, sound levels, etc.), inventory, and mobile camera work. In this paper, we first discuss the motivation for free-flying space robots. We then describe the development of the Smart SPHERES prototype, including avionics, software, and data communications. Finally, we present results of initial flight tests on-board the ISS.

  19. Smart SPHERES: A Telerobotic Free-Flyer for Intravehicular Activities in Space

    NASA Technical Reports Server (NTRS)

    Fong, Terrence; Micire, Mark J.; Morse, Ted; Park, Eric; Provencher, Chris

    2013-01-01

    Smart SPHERES is a prototype free-flying space robot based on the SPHERES platform. Smart SPHERES can be remotely operated by astronauts inside a spacecraft, or by mission controllers on the ground. We developed Smart SPHERES to perform a variety of intravehicular activities (IVA), such as operations inside the International Space Station (ISS). These IVA tasks include environmental monitoring surveys (radiation, sound levels, etc.), inventory, and mobile camera work. In this paper, we first discuss the motivation for free- flying space robots. We then describe the development of the Smart SPHERES prototype, including avionics, software, and data communications. Finally, we present results of initial flight tests on-board the ISS.

  20. Analyses of space environment effects on active fiber optic links orbited aboard the LDEF

    NASA Technical Reports Server (NTRS)

    Taylor, Edward W.; Monarski, T. W.; Berry, J. N.; Sanchez, A. D.; Padden, R. J.; Chapman, S. P.

    1993-01-01

    The results of the 'Preliminary Analysis of WL Experiment no. 701, Space Environment Effects on Operating Fiber Optic Systems,' is correlated with space simulated post retrieval terrestrial studies performed on the M0004 experiment. Temperature cycling measurements were performed on the active optical data links for the purpose of assessing link signal to noise ratio and bit error rate performance some 69 months following the experiment deployment in low Earth orbit. The early results indicate a high correlation between pre-orbit, orbit, and post-orbit functionality of the first known and longest space demonstration of operating fiber optic systems.

  1. Active control synthesis for flexible space structures excited by persistent disturbances

    NASA Technical Reports Server (NTRS)

    Wie, Bong; Gonzalez, Marcelo

    1990-01-01

    Both classical and state-space synthesis methods for active control of flexible space structures in the presence of persistent disturbances are presented. The methods exploit the so-called internal model principle for asymptotic disturbance rejection. A generic example of flexible space structures is used to illustrate the simplicity of the proposed design methodologies. The concept of a disturbance rejection filter dipole is introduced from a classical control viewpoint. It is shown that the proposed design methods will invariably make use of non-minimum-phase compensation for a class of noncolocated control problems. The need for tradeoffs between performance and parameter robustness is discussed.

  2. Propulsion/ASME Rocket-Based Combined Cycle Activities in the Advanced Space Transportation Program Office

    NASA Technical Reports Server (NTRS)

    Hueter, Uwe; Turner, James

    1998-01-01

    NASA's Office Of Aeronautics and Space Transportation Technology (OASTT) has establish three major coals. "The Three Pillars for Success". The Advanced Space Transportation Program Office (ASTP) at the NASA's Marshall Space Flight Center in Huntsville,Ala. focuses on future space transportation technologies under the "Access to Space" pillar. The Advanced Reusable Technologies (ART) Project, part of ASTP, focuses on the reusable technologies beyond those being pursued by X-33. The main activity over the past two and a half years has been on advancing the rocket-based combined cycle (RBCC) technologies. In June of last year, activities for reusable launch vehicle (RLV) airframe and propulsion technologies were initiated. These activities focus primarily on those technologies that support the year 2000 decision to determine the path this country will take for Space Shuttle and RLV. In February of this year, additional technology efforts in the reusable technologies were awarded. The RBCC effort that was completed early this year was the initial step leading to flight demonstrations of the technology for space launch vehicle propulsion. Aerojet, Boeing-Rocketdyne and Pratt & Whitney were selected for a two-year period to design, build and ground test their RBCC engine concepts. In addition, ASTROX, Pennsylvania State University (PSU) and University of Alabama in Huntsville also conducted supporting activities. The activity included ground testing of components (e.g., injectors, thrusters, ejectors and inlets) and integrated flowpaths. An area that has caused a large amount of difficulty in the testing efforts is the means of initiating the rocket combustion process. All three of the prime contractors above were using silane (SiH4) for ignition of the thrusters. This follows from the successful use of silane in the NASP program for scramjet ignition. However, difficulties were immediately encountered when silane (an 80/20 mixture of hydrogen/silane) was used for rocket

  3. Versatile in vivo regulation of tumor phenotypes by dCas9-mediated transcriptional perturbation

    PubMed Central

    Braun, Christian J.; Bruno, Peter M.; Horlbeck, Max A.; Gilbert, Luke A.; Weissman, Jonathan S.; Hemann, Michael T.

    2016-01-01

    Targeted transcriptional regulation is a powerful tool to study genetic mediators of cellular behavior. Here, we show that catalytically dead Cas9 (dCas9) targeted to genomic regions upstream or downstream of the transcription start site allows for specific and sustainable gene-expression level alterations in tumor cells in vitro and in syngeneic immune-competent mouse models. We used this approach for a high-coverage pooled gene-activation screen in vivo and discovered previously unidentified modulators of tumor growth and therapeutic response. Moreover, by using dCas9 linked to an activation domain, we can either enhance or suppress target gene expression simply by changing the genetic location of dCas9 binding relative to the transcription start site. We demonstrate that these directed changes in gene-transcription levels occur with minimal off-target effects. Our findings highlight the use of dCas9-mediated transcriptional regulation as a versatile tool to reproducibly interrogate tumor phenotypes in vivo. PMID:27325776

  4. No evidence of inhibition of horizontal gene transfer by CRISPR-Cas on evolutionary timescales.

    PubMed

    Gophna, Uri; Kristensen, David M; Wolf, Yuri I; Popa, Ovidiu; Drevet, Christine; Koonin, Eugene V

    2015-09-01

    The CRISPR (clustered, regularly, interspaced, short, palindromic repeats)-Cas (CRISPR-associated genes) systems of archaea and bacteria provide adaptive immunity against viruses and other selfish elements and are believed to curtail horizontal gene transfer (HGT). Limiting acquisition of new genetic material could be one of the sources of the fitness cost of CRISPR-Cas maintenance and one of the causes of the patchy distribution of CRISPR-Cas among bacteria, and across environments. We sought to test the hypothesis that the activity of CRISPR-Cas in microbes is negatively correlated with the extent of recent HGT. Using three independent measures of HGT, we found no significant dependence between the length of CRISPR arrays, which reflects the activity of the immune system, and the estimated number of recent HGT events. In contrast, we observed a significant negative dependence between the estimated extent of HGT and growth temperature of microbes, which could be explained by the lower genetic diversity in hotter environments. We hypothesize that the relevant events in the evolution of resistance to mobile elements and proclivity for HGT, to which CRISPR-Cas systems seem to substantially contribute, occur on the population scale rather than on the timescale of species evolution.

  5. Sacroillite tuberculeuse: à propos de deux cas

    PubMed Central

    Diallo, Ismaël; Zabsonré, Joëlle Tiendrébéogo; Kambou, Bénilde Marie Ange Tiemtoré; Sondo, Apoline Kongnimissom; Sagna, Yempabou; Ouédraogo, Dieu-Donné

    2016-01-01

    La sacroiliite tuberculeuse est rare et de diagnostic difficile. Les auteurs rapportent deux cas. Il s'agissait dans le premier cas d'une patiente de 40 ans ayant une infection à VIH ; le diagnostic a été histologique après une biopsie chirurgicale. Le second cas a concerné un patient de 25 ans vivant en milieu carcéral chez qui le diagnostic a été établi sur la base des arguments cliniques, biologiques, radiologiques et l'efficacité du traitement ; l'intradermoréaction à la tuberculine était phlycténulaire. Le scanner a été indispensable au diagnostic lésionnel en montrant une érosion des berges et des abcès des parties molles. Le traitement a été médical et a fait appel aux antituberculeux. PMID:28292032

  6. Interference Mitigation Technique Using Active Spaceborne Sensor Antenna in EESS (Active) and Space Research Service (Active) for Use in 500 MHz Bandwidth Near 9.6 GHz

    NASA Technical Reports Server (NTRS)

    Huneycutt, Bryan L.

    2005-01-01

    This document presents an interference mitigation technique using the active spaceborne sensor SAR3 antenna in the Earth Exploration-Satellite Service (active) and Space Research Service (active) for use in a 500 MHz bandwidth near 9.6 GHz. The purpose of the document is present antenna designs which offer lower sidelobes and faster rolloff in the sidelobes which in turn mitigates the interference to other services from the EESS (active) and SRS (active) sensors.

  7. Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28.

    PubMed

    Smargon, Aaron A; Cox, David B T; Pyzocha, Neena K; Zheng, Kaijie; Slaymaker, Ian M; Gootenberg, Jonathan S; Abudayyeh, Omar A; Essletzbichler, Patrick; Shmakov, Sergey; Makarova, Kira S; Koonin, Eugene V; Zhang, Feng

    2017-02-16

    CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 and Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.

  8. Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS.

    PubMed

    Pul, Umit; Wurm, Reinhild; Arslan, Zihni; Geissen, René; Hofmann, Nina; Wagner, Rolf

    2010-03-01

    Inheritable bacterial defence systems against phage infection and foreign DNA, termed CRISPR (clustered regularly interspaced short palindromic repeats), consist of cas protein genes and repeat arrays interspaced with sequences originating from invaders. The Cas proteins together with processed small spacer-repeat transcripts (crRNAs) cause degradation of penetrated foreign DNA by unknown mechanisms. Here, we have characterized previously unidentified promoters of the Escherichia coli CRISPR arrays and cas protein genes. Transcription of precursor crRNA is directed by a promoter located within the CRISPR leader. A second promoter, directing cas gene transcription, is located upstream of the genes encoding proteins of the Cascade complex. Furthermore, we demonstrate that the DNA-binding protein H-NS is involved in silencing the CRISPR-cas promoters, resulting in cryptic Cas protein expression. Our results demonstrate an active involvement of H-NS in the induction of the CRISPR-cas system and suggest a potential link between two prokaryotic defence systems against foreign DNA.

  9. Evolution and classification of the CRISPR-Cas systems

    PubMed Central

    S. Makarova, Kira; H. Haft, Daniel; Barrangou, Rodolphe; J. J. Brouns, Stan; Charpentier, Emmanuelle; Horvath, Philippe; Moineau, Sylvain; J. M. Mojica, Francisco; I. Wolf, Yuri; Yakunin, Alexander F.; van der Oost, John; V. Koonin, Eugene

    2012-01-01

    The CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR–Cas systems and Cas proteins. Three major types of CRISPR–Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR–Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a `polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR–cas loci. PMID:21552286

  10. SnapShot: Class 1 CRISPR-Cas Systems.

    PubMed

    Makarova, Kira S; Zhang, Feng; Koonin, Eugene V

    2017-02-23

    Class 1 CRISPR-Cas systems are characterized by effector modules consisting of multiple subunits. Class 1 systems comprise about 90% of all CRISPR-Cas loci identified in bacteria and archaea and can target both DNA and RNA.

  11. CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

    PubMed

    Yuen, Kit-San; Chan, Chi-Ping; Wong, Nok-Hei Mickey; Ho, Chau-Ha; Ho, Ting-Hin; Lei, Ting; Deng, Wen; Tsao, Sai Wah; Chen, Honglin; Kok, Kin-Hang; Jin, Dong-Yan

    2015-03-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.

  12. Biased allosteric modulation at the CaS receptor engendered by structurally diverse calcimimetics

    PubMed Central

    Cook, A E; Mistry, S N; Gregory, K J; Furness, S G B; Sexton, P M; Scammells, P J; Conigrave, A D; Christopoulos, A; Leach, K

    2015-01-01

    BACKGROUND AND PURPOSE Clinical use of cinacalcet in hyperparathyroidism is complicated by its tendency to induce hypocalcaemia, arising partly from activation of calcium-sensing receptors (CaS receptors) in the thyroid and stimulation of calcitonin release. CaS receptor allosteric modulators that selectively bias signalling towards pathways that mediate desired effects [e.g. parathyroid hormone (PTH) suppression] rather than those mediating undesirable effects (e.g. elevated serum calcitonin), may offer better therapies. EXPERIMENTAL APPROACH We characterized the ligand-biased profile of novel calcimimetics in HEK293 cells stably expressing human CaS receptors, by monitoring intracellular calcium (Ca2+i) mobilization, inositol phosphate (IP)1 accumulation, ERK1/2 phosphorylation (pERK1/2) and receptor expression. KEY RESULTS Phenylalkylamine calcimimetics were biased towards allosteric modulation of Ca2+i mobilization and IP1 accumulation. S,R-calcimimetic B was biased only towards IP1 accumulation. R,R-calcimimetic B and AC-265347 were biased towards IP1 accumulation and pERK1/2. Nor-calcimimetic B was unbiased. In contrast to phenylalkylamines and calcimimetic B analogues, AC-265347 did not promote trafficking of a loss-of-expression, naturally occurring, CaS receptor mutation (G670E). CONCLUSIONS AND IMPLICATIONS The ability of R,R-calcimimetic B and AC-265347 to bias signalling towards pERK1/2 and IP1 accumulation may explain their suppression of PTH levels in vivo at concentrations that have no effect on serum calcitonin levels. The demonstration that AC-265347 promotes CaS receptor receptor signalling, but not trafficking reveals a novel profile of ligand-biased modulation at CaS receptors The identification of allosteric modulators that bias CaS receptor signalling towards distinct intracellular pathways provides an opportunity to develop desirable biased signalling profiles in vivo for mediating selective physiological responses. PMID:25220431

  13. Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9.

    PubMed

    Mougiakos, Ioannis; Bosma, Elleke F; Weenink, Koen; Vossen, Eric; Goijvaerts, Kirsten; van der Oost, John; van Kranenburg, Richard

    2017-02-16

    Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e., Bacillus smithii, including a gene deletion, gene knockout via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42 °C, and we employ the wide temperature growth range of B. smithii as an induction system for spCas9 expression. Homologous recombination with plasmid-borne editing templates is performed at 45-55 °C, when spCas9 is inactive. Subsequent transfer to 37 °C allows for counterselection through production of active spCas9, which introduces lethal double-stranded DNA breaks to the nonedited cells. The developed method takes 4 days with 90, 100, and 20% efficiencies for gene deletion, knockout, and insertion, respectively. The major advantage of our system is the limited requirement for genetic parts: only one plasmid, one selectable marker, and a promoter are needed, and the promoter does not need to be inducible or well-characterized. Hence, it can be easily applied for genome editing purposes in both mesophilic and thermophilic nonmodel organisms with a limited genetic toolbox and ability to grow at, or tolerate, temperatures of 37 and at or above 42 °C.

  14. The CRISPR-Cas system - from bacterial immunity to genome engineering.

    PubMed

    Czarnek, Maria; Bereta, Joanna

    2016-09-01

    Precise and efficient genome modifications present a great value in attempts to comprehend the roles of particular genes and other genetic elements in biological processes as well as in various pathologies. In recent years novel methods of genome modification known as genome editing, which utilize so called "programmable" nucleases, came into use. A true revolution in genome editing has been brought about by the introduction of the CRISP-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) system, in which one of such nucleases, i.e. Cas9, plays a major role. This system is based on the elements of the bacterial and archaeal mechanism responsible for acquired immunity against phage infections and transfer of foreign genetic material. Microorganisms incorporate fragments of foreign DNA into CRISPR loci present in their genomes, which enables fast recognition and elimination of future infections. There are several types of CRISPR-Cas systems among prokaryotes but only elements of CRISPR type II are employed in genome engineering. CRISPR-Cas type II utilizes small RNA molecules (crRNA and tracrRNA) to precisely direct the effector nuclease - Cas9 - to a specific site in the genome, i.e. to the sequence complementary to crRNA. Cas9 may be used to: (i) introduce stable changes into genomes e.g. in the process of generation of knock-out and knock-in animals and cell lines, (ii) activate or silence the expression of a gene of interest, and (iii) visualize specific sites in genomes of living cells. The CRISPR-Cas-based tools have been successfully employed for generation of animal and cell models of a number of diseases, e.g. specific types of cancer. In the future, the genome editing by programmable nucleases may find wide application in medicine e.g. in the therapies of certain diseases of genetic origin and in the therapy of HIV-infected patients.

  15. Streamlined design and self reliant hardware for active control of precision space structures

    NASA Technical Reports Server (NTRS)

    Hyland, David C.; King, James A.; Phillips, Douglas J.

    1994-01-01

    Precision space structures may require active vibration control to satisfy critical performance requirements relating to line-of-sight pointing accuracy and the maintenance of precise, internal alignments. In order for vibration control concepts to become operational, it is necessary that their benefits be practically demonstrated in large scale ground-based experiments. A unique opportunity to carry out such demonstrations on a wide variety of experimental testbeds was provided by the NASA Control-Structure Integration (CSI) Guest Investigator (GI) Program. This report surveys the experimental results achieved by the Harris Corporation GI team on both Phases 1 and 2 of the program and provides a detailed description of Phase 2 activities. The Phase 1 results illustrated the effectiveness of active vibration control for space structures and demonstrated a systematic methodology for control design, implementation test. In Phase 2, this methodology was significantly streamlined to yield an on-site, single session design/test capability. Moreover, the Phase 2 research on adaptive neural control techniques made significant progress toward fully automated, self-reliant space structure control systems. As a further thrust toward productized, self-contained vibration control systems, the Harris Phase II activity concluded with experimental demonstration of new vibration isolation hardware suitable for a wide range of space-flight and ground-based commercial applications.The CSI GI Program Phase 1 activity was conducted under contract NASA1-18872, and the Phase 2 activity was conducted under NASA1-19372.

  16. Effects of repeated simulated removal activities on feral swine movements and space use

    USGS Publications Warehouse

    Fischer, Justin W.; McMurtry , Dan; Blass, Chad R.; Walter, William D.; Beringer, Jeff; VerCauterren, Kurt C.

    2016-01-01

    Abundance and distribution of feral swine (Sus scrofa) in the USA have increased dramatically during the last 30 years. Effective measures are needed to control and eradicate feral swine populations without displacing animals over wider areas. Our objective was to investigate effects of repeated simulated removal activities on feral swine movements and space use. We analyzed location data from 21 feral swine that we fitted with Global Positioning System harnesses in southern MO, USA. Various removal activities were applied over time to eight feral swine before lethal removal, including trapped-and-released, chased with dogs, chased with hunter, and chased with helicopter. We found that core space-use areas were reduced following the first removal activity, whereas overall space-use areas and diurnal movement distances increased following the second removal activity. Mean geographic centroid shifts did not differ between pre- and post-periods for either the first or second removal activities. Our information on feral swine movements and space use precipitated by human removal activities, such as hunting, trapping, and chasing with dogs, helps fill a knowledge void and will aid wildlife managers. Strategies to optimize management are needed to reduce feral swine populations while preventing enlarged home ranges and displacing individuals, which could lead to increased disease transmission risk and human-feral swine conflict in adjacent areas.

  17. A review of UK space activity and historiography, 1957-2007

    NASA Astrophysics Data System (ADS)

    Millard, Douglas

    2010-04-01

    In over 50 years the United Kingdom has designed, built, launched, operated or otherwise contributed to hundreds of spacecraft and space missions. Its scientists, engineers and officials have carved centres of astronautical excellence around the country, participated in a great number of international space programmes and missions and played a leading role in the establishment of the world's main pan-national space agency (ESA) and its two precursors, the European Launcher Development Organisation (ELDO) and the European Space Research Organization (ESRO). With its Skylark sounding rocket launch of November 1957 the UK was one of the first nations to gather new scientific data as part of the International Geophysical Year. Fifty years on, the UK is an enthusiastic supporter of the Global Exploration Strategy with major commitments to future missions to the moon and to the Mars that exploit the nation's expertise in small satellite and planetary robot technology. And while such mission involvement takes UK space technologies out into the solar system as never before the nation continues to excel in Earth orbit with its development and manufacture of large, increasingly powerful telecommunications satellites. The UK's space heritage and its ongoing and directed activities are rich and productive. And yet—the representation of UK space endeavour is all too often skewed—misleading and unduly pejorative: '…British space…more romance than reality.' Why does such partisan commentary occur and why has such an attitude prevailed for so long? This paper seeks some answers by reviewing UK space activity and its historiography in the wider and global context of astronautics between 1957 and 2007. In Praise of…the British Space Programme, The Guardian Newspaper, March 4th, 2008.

  18. Density-Dependent Spacing Behaviour and Activity Budget in Pregnant, Domestic Goats (Capra hircus).

    PubMed

    Vas, Judit; Andersen, Inger Lise

    2015-01-01

    Very little is known about the spacing behaviour in social groups of domestic goats (Capra hircus) in the farm environment. In this experiment, we studied interindividual distances, movement patterns and activity budgets in pregnant goats housed at three different densities. Norwegian dairy goats were kept in stable social groups of six animals throughout pregnancy at 1, 2 or 3 m2 per individual and their spacing behaviours (i.e., distance travelled, nearest and furthest neighbour distance) and activity budgets (e.g., resting, feeding, social activities) were monitored. Observations were made in the first, second and last thirds of pregnancy in the mornings, at noon and in the afternoons of each of these phases (4.5 hours per observation period). The findings show that goats held at animal densities of 2 and 3 m2 moved longer distances when they had more space per animal and kept larger nearest and furthest neighbour distances when compared to the 1 m2 per animal density. Less feeding activity was observed at the high animal density compared to the medium and low density treatments. The phase of gestation also had an impact on almost all behavioural variables. Closer to parturition, animals moved further distances and the increase in nearest and furthest neighbour distance was more pronounced at the lower animal densities. During the last period of gestation, goats spent less time feeding and more on resting, social behaviours and engaging in other various activities. Our data suggest that more space per goat is needed for goats closer to parturition than in the early gestation phase. We concluded that in goats spacing behaviour is density-dependent and changes with stages of pregnancy and activities. Finally, the lower density allowed animals to express individual preferences regarding spacing behaviour which is important in ensuring good welfare in a farming situation.

  19. Density-Dependent Spacing Behaviour and Activity Budget in Pregnant, Domestic Goats (Capra hircus)

    PubMed Central

    Vas, Judit; Andersen, Inger Lise

    2015-01-01

    Very little is known about the spacing behaviour in social groups of domestic goats (Capra hircus) in the farm environment. In this experiment, we studied interindividual distances, movement patterns and activity budgets in pregnant goats housed at three different densities. Norwegian dairy goats were kept in stable social groups of six animals throughout pregnancy at 1, 2 or 3 m2 per individual and their spacing behaviours (i.e. distance travelled, nearest and furthest neighbour distance) and activity budgets (e.g. resting, feeding, social activities) were monitored. Observations were made in the first, second and last thirds of pregnancy in the mornings, at noon and in the afternoons of each of these phases (4.5 hours per observation period). The findings show that goats held at animal densities of 2 and 3 m2 moved longer distances when they had more space per animal and kept larger nearest and furthest neighbour distances when compared to the 1 m2 per animal density. Less feeding activity was observed at the high animal density compared to the medium and low density treatments. The phase of gestation also had an impact on almost all behavioural variables. Closer to parturition, animals moved further distances and the increase in nearest and furthest neighbour distance was more pronounced at the lower animal densities. During the last period of gestation, goats spent less time feeding and more on resting, social behaviours and engaging in other various activities. Our data suggest that more space per goat is needed for goats closer to parturition than in the early gestation phase. We concluded that in goats spacing behaviour is density-dependent and changes with stages of pregnancy and activities. Finally, the lower density allowed animals to express individual preferences regarding spacing behaviour which is important in ensuring good welfare in a farming situation. PMID:26657240

  20. CRISPR-Cas immunity in prokaryotes.

    PubMed

    Marraffini, Luciano A

    2015-10-01

    Prokaryotic organisms are threatened by a large array of viruses and have developed numerous defence strategies. Among these, only clustered, regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity against foreign elements. Upon viral injection, a small sequence of the viral genome, known as a spacer, is integrated into the CRISPR locus to immunize the host cell. Spacers are transcribed into small RNA guides that direct the cleavage of the viral DNA by Cas nucleases. Immunization through spacer acquisition enables a unique form of evolution whereby a population not only rapidly acquires resistance to its predators but also passes this resistance mechanism vertically to its progeny.

  1. The law applicable to the use of space for commercial activities

    NASA Technical Reports Server (NTRS)

    Hosenball, S. N.

    1983-01-01

    The general principles of space law that have an impact on commercial space activities are discussed. The Outer Space Treaty guaranteed the right of private enterprise in space, with jurisdiction over the participating parties residing in the country of origin. The liability for damages caused to a third party is also assigned to the country of origin. Government consent is necessary in the U.S. before a private firm is permitted to launch an object into space, with the relevant statute sections being part of the Arms Export Control Act; launches are legally treated as exports. FAA regulations define the safe area and flight conditions that must be satisfied for a private launch, although NASA, in the 1958 act which formed the agency, potentialy has the power to regulate space launch activities. The DoD must be notified of any launches in order to notify the U.S.S.R., filings must be made with the Bureau of Alcohol, Tobacco, and Firearms, and fees must be paid to the IRS. It is presently U.S. government policy to encourage and facilitate private sector development of commercial launch services.

  2. Task-specific stability in muscle activation space during unintentional movements.

    PubMed

    Falaki, Ali; Towhidkhah, Farzad; Zhou, Tao; Latash, Mark L

    2014-11-01

    We used robot-generated perturbations applied during position-holding tasks to explore stability of induced unintentional movements in a multidimensional space of muscle activations. Healthy subjects held the handle of a robot against a constant bias force and were instructed not to interfere with hand movements produced by changes in the external force. Transient force changes were applied leading to handle displacement away from the initial position and then back toward the initial position. Intertrial variance in the space of muscle modes (eigenvectors in the muscle activations space) was quantified within two subspaces, corresponding to unchanged handle coordinate and to changes in the handle coordinate. Most variance was confined to the former subspace in each of the three phases of movement, the initial steady state, the intermediate position, and the final steady state. The same result was found when the changes in muscle activation were analyzed between the initial and final steady states. Changes in the dwell time between the perturbation force application and removal led to different final hand locations undershooting the initial position. The magnitude of the undershot scaled with the dwell time, while the structure of variance in the muscle activation space did not depend on the dwell time. We conclude that stability of the hand coordinate is ensured during both intentional and unintentional actions via similar mechanisms. Relative equifinality in the external space after transient perturbations may be associated with varying states in the redundant space of muscle activations. The results fit a hierarchical scheme for the control of voluntary movements with referent configurations and redundant mapping between the levels of the hierarchy.

  3. Engineering of temperature- and light-switchable Cas9 variants

    PubMed Central

    Richter, Florian; Fonfara, Ines; Bouazza, Boris; Schumacher, Charlotte Helene; Bratovič, Majda; Charpentier, Emmanuelle; Möglich, Andreas

    2016-01-01

    Sensory photoreceptors have enabled non-invasive and spatiotemporal control of numerous biological processes. Photoreceptor engineering has expanded the repertoire beyond natural receptors, but to date no generally applicable strategy exists towards constructing light-regulated protein actuators of arbitrary function. We hence explored whether the homodimeric Rhodobacter sphaeroides light-oxygen-voltage (LOV) domain (RsLOV) that dissociates upon blue-light exposure can confer light sensitivity onto effector proteins, via a mechanism of light-induced functional site release. We chose the RNA-guided programmable DNA endonuclease Cas9 as proof-of-principle effector, and constructed a comprehensive library of RsLOV inserted throughout the Cas9 protein. Screening with a high-throughput assay based on transcriptional repression in Escherichia coli yielded paRC9, a moderately light-activatable variant. As domain insertion can lead to protein destabilization, we also screened the library for temperature-sensitive variants and isolated tsRC9, a variant with robust activity at 29°C but negligible activity at 37°C. Biochemical assays confirmed temperature-dependent DNA cleavage and binding for tsRC9, but indicated that the light sensitivity of paRC9 is specific to the cellular setting. Using tsRC9, the first temperature-sensitive Cas9 variant, we demonstrate temperature-dependent transcriptional control over ectopic and endogenous genetic loci. Taken together, RsLOV can confer light sensitivity onto an unrelated effector; unexpectedly, the same LOV domain can also impart strong temperature sensitivity. PMID:27744350

  4. Quantum-classical transition and quantum activation of ratchet currents in the parameter space.

    PubMed

    Beims, M W; Schlesinger, M; Manchein, C; Celestino, A; Pernice, A; Strunz, W T

    2015-05-01

    The quantum ratchet current is studied in the parameter space of the dissipative kicked rotor model coupled to a zero-temperature quantum environment. We show that vacuum fluctuations blur the generic isoperiodic stable structures found in the classical case. Such structures tend to survive when a measure of statistical dependence between the quantum and classical currents are displayed in the parameter space. In addition, we show that quantum fluctuations can be used to overcome transport barriers in the phase space. Related quantum ratchet current activation regions are spotted in the parameter space. Results are discussed based on quantum, semiclassical, and classical calculations. While the semiclassical dynamics involves vacuum fluctuations, the classical map is driven by thermal noise.

  5. NASA Crew Personal Active Dosimeters (CPADs): Leveraging Novel Terrestrial Personal Radiation Monitoring Capabilities for Space Exploration

    NASA Technical Reports Server (NTRS)

    Leitgab, Martin; Semones, Edward; Lee, Kerry

    2016-01-01

    The NASA Space Radiation Analysis Group (SRAG) is developing novel Crew Personal Active Dosimeters (CAPDs) for upcoming crewed space exploration missions and beyond. To reduce the resource footprint of the project a COTS dosimeter base is used for the development of CPADs. This base was identified from evaluations of existing COTS personal dosimeters against the concept of operations of future crewed missions and tests against detection requirements for radiation characteristic of the space environment. CPADs exploit operations efficiencies from novel features for space flight personal dosimeters such as real-time dose feedback, and autonomous measuring and data transmission capabilities. Preliminary CPAD design, results of radiation testing and aspects of operational integration will be presented.

  6. Green spaces and General Health: Roles of mental health status, social support, and physical activity.

    PubMed

    Dadvand, Payam; Bartoll, Xavier; Basagaña, Xavier; Dalmau-Bueno, Albert; Martinez, David; Ambros, Albert; Cirach, Marta; Triguero-Mas, Margarita; Gascon, Mireia; Borrell, Carme; Nieuwenhuijsen, Mark J

    2016-05-01

    Green spaces are associated with improved health, but little is known about mechanisms underlying such association. We aimed to assess the association between greenness exposure and subjective general health (SGH) and to evaluate mental health status, social support, and physical activity as mediators of this association. This cross-sectional study was based on a population-based sample of 3461 adults residing in Barcelona, Spain (2011). We characterized outcome and mediators using the Health Survey of Barcelona. Objective and subjective residential proximity to green spaces and residential surrounding greenness were used to characterize greenness exposure. We followed Baron and Kenny's framework to establish the mediation roles and we further quantified the relative contribution of each mediator. Residential surrounding greenness and subjective residential proximity to green spaces were associated with better SGH. We found indications for mediation of these associations by mental health status, perceived social support, and to less extent, by physical activity. These mediators altogether could explain about half of the surrounding greenness association and one-third of the association for subjective proximity to green spaces. We observed indications that mental health and perceived social support might be more relevant for men and those younger than 65years. The results for objective residential proximity to green spaces were not conclusive. In conclusion, our observed association between SGH and greenness exposure was mediated, in part, by mental health status, enhanced social support, and physical activity. There might be age and sex variations in these mediation roles.

  7. CRISPR-Cas9 nuclear dynamics and target recognition in living cells

    PubMed Central

    Ma, Hanhui; Tu, Li-Chun; Zhang, Shaojie; Grunwald, David

    2016-01-01

    The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)–associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9–guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from >3 h to as low as <2 min in a nucleotide identity- and position-dependent manner. We further show that the duration of target residence correlates with cleavage activity. These results reveal that CRISPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time. PMID:27551060

  8. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery

    PubMed Central

    Lin, Steven; Staahl, Brett T; Alla, Ravi K; Doudna, Jennifer A

    2014-01-01

    The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells. DOI: http://dx.doi.org/10.7554/eLife.04766.001 PMID:25497837

  9. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.

    PubMed

    Lin, Steven; Staahl, Brett T; Alla, Ravi K; Doudna, Jennifer A

    2014-12-15

    The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

  10. A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation.

    PubMed

    Lowder, Levi G; Zhang, Dengwei; Baltes, Nicholas J; Paul, Joseph W; Tang, Xu; Zheng, Xuelian; Voytas, Daniel F; Hsieh, Tzung-Fu; Zhang, Yong; Qi, Yiping

    2015-10-01

    The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.

  11. Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering.

    PubMed

    Ui-Tei, Kumiko; Maruyama, Shohei; Nakano, Yuko

    2017-01-26

    Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

  12. Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis.

    PubMed

    Guo, Xiaogang; Zhang, Tiejun; Hu, Zheng; Zhang, Yanqi; Shi, Zhaoying; Wang, Qinhu; Cui, Yan; Wang, Fengqin; Zhao, Hui; Chen, Yonglong

    2014-02-01

    For the emerging amphibian genetic model Xenopus tropicalis targeted gene disruption is dependent on zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), which require either complex design and selection or laborious construction. Thus, easy and efficient genome editing tools are still highly desirable for this species. Here, we report that RNA-guided Cas9 nuclease resulted in precise targeted gene disruption in all ten X. tropicalis genes that we analyzed, with efficiencies above 45% and readily up to 100%. Systematic point mutation analyses in two loci revealed that perfect matches between the spacer and the protospacer sequences proximal to the protospacer adjacent motif (PAM) were essential for Cas9 to cleave the target sites in the X. tropicalis genome. Further study showed that the Cas9 system could serve as an efficient tool for multiplexed genome engineering in Xenopus embryos. Analysis of the disruption of two genes, ptf1a/p48 and tyrosinase, indicated that Cas9-mediated gene targeting can facilitate direct phenotypic assessment in X. tropicalis embryos. Finally, five founder frogs from targeting of either elastase-T1, elastase-T2 or tyrosinase showed highly efficient transmission of targeted mutations into F1 embryos. Together, our data demonstrate that the Cas9 system is an easy, efficient and reliable tool for multiplex genome editing in X. tropicalis.

  13. ActiveSpaces on the grid: The construction of advanced visualization and interaction environments

    SciTech Connect

    Childers, L.; Disz, T.; Hereld, M.; Hudson, R.; Judson, I.; Olson, R.; Papka, M. E.; Paris, J.; Stevens, R.

    2000-07-24

    The Futures Lab group at Argonne National Laboratory and the University of Chicago are designing, building, and evaluating a new type of interactive computing environment that couples in a deep way the concepts of direct manipulation found in virtual reality with the richness and variety of interactive devices found in ubiquitous computing. This environment provides the interactivity and collaboration support of teleimmersive environments with the exibility and availability of desktop collaboration tools. The authors call these environments ActiveSpaces. An ActiveSpace is a physical domain that has been augmented with multiscale multiscreen displays, environment-specific and device-specific sensors, body and object trackers, human-input and instrument-input interfaces, streaming audio and video capture devices, and force feedback devices--and has then been connected to other such spaces via the Grid.

  14. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.

    PubMed

    Pattanayak, Vikram; Lin, Steven; Guilinger, John P; Ma, Enbo; Doudna, Jennifer A; Liu, David R

    2013-09-01

    The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

  15. Boosting plant immunity with CRISPR/Cas.

    PubMed

    Chaparro-Garcia, Angela; Kamoun, Sophien; Nekrasov, Vladimir

    2015-11-19

    CRISPR/Cas has recently been transferred to plants to make them resistant to geminiviruses, a damaging family of DNA viruses. We discuss the potential and the limitations of this method.See related Research: http://www.genomebiology.com/2015/16/1/238.

  16. Using the CAS Standards in Assessment Projects

    ERIC Educational Resources Information Center

    Dean, Laura A.

    2013-01-01

    This chapter provides an overview of the use of professional standards of practice in assessment and of the Council for the Advancement of Standards in Higher Education (CAS). It outlines a model for conducting program self-studies and discusses the importance of implementing change based on assessment results.

  17. Effects of Using a Computer Algebra System (CAS) on Junior College Students' Attitudes towards CAS and Achievement in Mathematics

    ERIC Educational Resources Information Center

    Leng, Ng Wee; Choo, Kwee Tiow; Soon, Lau Hock; Yi-Huak, Koh; Sun, Yap Yew

    2005-01-01

    This study examines the effects of using Texas Instruments' Voyage 200 calculator (V200), a graphing calculator with a built-in computer algebra system (CAS), on attitudes towards CAS and achievement in mathematics of junior college students (17 year olds). Students' attitudes towards CAS were examined using a 40-item Likert-type instrument…

  18. Young Children's Literacy in the Activity Space of the Library: A Geosemiotic Investigation

    ERIC Educational Resources Information Center

    Nichols, Sue

    2011-01-01

    An ecological approach, emphasizing the importance of understanding multiple contexts for learning, underpins this study of libraries as activity spaces for young children's literacy participation. Five libraries serving a diversity of communities were the subject of ethnographic investigation incorporating participant observation, visual…

  19. Looking Inside and Out: Perceptions of Physical Activity in Childcare Spaces

    ERIC Educational Resources Information Center

    Weinberger, Nanci; Butler, Allison G.; Schumacher, Phyllis

    2014-01-01

    This study addresses the need to better understand how providers' perceptions of indoor and outdoor childcare settings can set the stage for arranging play spaces to optimise children's moderate-to-vigorous physical play. Childcare providers' perceptions of the level of physical activity, safety, and quality that children experience…

  20. Adaptation of neuromuscular activation patterns during treadmill walking after long-duration space flight

    NASA Astrophysics Data System (ADS)

    Layne, C. S.; Lange, G. W.; Pruett, C. J.; McDonald, P. V.; Merkle, L. A.; Mulavara, A. P.; Smith, S. L.; Kozlovskaya, I. B.; Bloomberg, J. J.

    The precise neuromuscular control needed for optimal locomotion, particularly around heel strike and toe off, is known to be compromised after short duration (8- to 15-day) space flight. We hypothesized here that longer exposure to weightlessness would result in maladaptive neuromuscular activation during postflight treadmill walking. We also hypothesized that space flight would affect the ability of the sensory-motor control system to generate adaptive neuromuscular activation patterns in response to changes in visual target distance during postflight treadmill walking. Seven crewmembers, who completed 3- to 6-month missions, walked on a motorized treadmill while visually fixating on a target placed 30 cm (NEAR) or 2 m (FAR) from the subject's eyes. Electronic foot switch data and surface electromyography were collected from selected muscles of the right lower limb. Results indicate that the phasic features of neuromuscular activation were moderately affected and the relative amplitude of activity in the tibialis anterior and rectus femoris around toe off changed after space flight. Changes also were evident after space flight in how these muscles adapted to the shift in visual target distance.

  1. "Go Be a Writer": Intra-Activity with Materials, Time and Space in Literacy Learning

    ERIC Educational Resources Information Center

    Kuby, Candace R.; Rucker, Tara Gutshall; Kirchhofer, Jessica M.

    2015-01-01

    This article is based on research in a United States second-grade classroom during a multimodal literacy workshop. Observing students working with tissue paper, foam board, string, pipe cleaners and other materials, we asked how is intra-activity with materials, time and space influencing literacy learning in Room 203? While the research…

  2. [Analysis of possible causes activation a stomach and pancreas excretory and incretory function after completion of space flight on the international space station].

    PubMed

    Afonin, B V

    2013-01-01

    The research excretory and incretory of activity of a stomach and pancreas is carried out at astronauts in the early period after completion of space flights of various duration. It is shown, that the increase of the contents of gastric and pancreatic enzymes and hormones (insulin and C-peptide) in blood reflects increased excretory and incretory activity of organs of gastroduodenal area which arises in weightlessness. The complex of countermeasures, which prevent ingress of subjects, infected by Helicobacter pylori in space flight crew, excluded participation of this microorganism in the mechanism of increase of secretory activity of a stomach. The absence of interrelation between increase of secretory activity of gastroduodenal area organs and space flights' duration has allowed to exclude the hypokinetic mechanism which determined by duration of stay in weightlessness. It was shown that after the end of space flights the increase ofbasal excretory activity of organs of gastroduodenal area occurs simultaneously with increase of a fasting insulin secretion. The changes in gastroduodenal area organs revealed after space flights were are compared to similar changes received in ground-based experiments, simulating hemodynamic reorganization in venous system of abdominal cavity, arising in weightlessness. The conclusion is made, that the basic mechanism of changes of a functional condition of digestive system in space flights, is determined by reorganization venous hemodynamic in abdominal cavity organs reproduced in ground experiments. Increase insulin and C-peptide after space flights are considered as hormonal component of this hemodynamic mechanism.

  3. CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA

    PubMed Central

    Zhou, Jianting; Wu, Ronghai; Xue, Xiaoli; Qin, Zhongjun

    2016-01-01

    Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated ‘CasHRA (Cas9-facilitated Homologous Recombination Assembly)’ that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 (Minimal Genome of Escherichia coli) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions. PMID:27220470

  4. Exploiting the CRISPR/Cas9 PAM Constraint for Single-Nucleotide Resolution Interventions

    PubMed Central

    Li, Yi; Mendiratta, Saurabh; Ehrhardt, Kristina; Kashyap, Neha; White, Michael A.; Bleris, Leonidas

    2016-01-01

    CRISPR/Cas9 is an enabling RNA-guided technology for genome targeting and engineering. An acute DNA binding constraint of the Cas9 protein is the Protospacer Adjacent Motif (PAM). Here we demonstrate that the PAM requirement can be exploited to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on the wild-type alleles. Specifically, we target the heterozygous G13A activating mutation of KRAS in colorectal cancer cells and we show reversal of drug resistance to a MEK small-molecule inhibitor. Our study introduces a new paradigm in genome editing and therapeutic targeting via the use of gRNA to guide Cas9 to a desired protospacer adjacent motif. PMID:26788852

  5. CRISPR-Cas and restriction-modification systems are compatible and increase phage resistance.

    PubMed

    Dupuis, Marie-Ève; Villion, Manuela; Magadán, Alfonso H; Moineau, Sylvain

    2013-01-01

    Bacteria have developed a set of barriers to protect themselves against invaders such as phage and plasmid nucleic acids. Different prokaryotic defence systems exist and at least two of them directly target the incoming DNA: restriction-modification (R-M) and CRISPR-Cas systems. On their own, they are imperfect barriers to invasion by foreign DNA. Here, we show that R-M and CRISPR-Cas systems are compatible and act together to increase the overall phage resistance of a bacterial cell by cleaving their respective target sites. Furthermore, we show that the specific methylation of phage DNA does not impair CRISPR-Cas acquisition or interference activities. Taken altogether, both mechanisms can be leveraged to decrease phage contaminations in processes relying on bacterial growth and/or fermentation.

  6. New clues on the regulation of the CRISPR-Cas immune system.

    PubMed

    Lundgren, Magnus

    2015-01-01

    Research into the CRISPR-Cas immune system of prokaryotes is progressing at a tremendous pace given both its important biological function and its role as a source of new genetic tools. However, a few areas of the field have remained largely unaddressed. A recent report provides information on one such overlooked area: how the cell regulates the CRISPR-Cas immune system. The processes, despite their importance, have remained illusive. In Pectobacterium atrosepticum regulation is, perhaps surprisingly, based on metabolic factors responding to glucose levels in the cell. Regulators include both activators and repressors of cas gene expression. It remains an open question why and how this regulatory system have evolved, and if it is a typical example of how CRISPR-as systems are regulated or not.

  7. New clues on the regulation of the CRISPR-Cas immune system

    PubMed Central

    Lundgren, Magnus

    2015-01-01

    Research into the CRISPR-Cas immune system of prokaryotes is progressing at a tremendous pace given both its important biological function and its role as a source of new genetic tools. However, a few areas of the field have remained largely unaddressed. A recent report provides information on one such overlooked area: how the cell regulates the CRISPR-Cas immune system. The processes, despite their importance, have remained illusive. In Pectobacterium atrosepticum regulation is, perhaps surprisingly, based on metabolic factors responding to glucose levels in the cell. Regulators include both activators and repressors of cas gene expression. It remains an open question why and how this regulatory system have evolved, and if it is a typical example of how CRISPR-as systems are regulated or not. PMID:26942048

  8. Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements.

    PubMed

    Karvelis, Tautvydas; Gasiunas, Giedrius; Young, Joshua; Bigelyte, Greta; Silanskas, Arunas; Cigan, Mark; Siksnys, Virginijus

    2015-11-19

    To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid libraries containing a randomized PAM as a function of Cas9-guide RNA complex concentration. Using this method, we accurately reproduce the canonical PAM preferences for Streptococcus pyogenes, Streptococcus thermophilus CRISPR3 (Sth3), and CRISPR1 (Sth1). Additionally, PAM and sgRNA solutions for a novel Cas9 protein from Brevibacillus laterosporus are provided by the assay and are demonstrated to support functional activity in vitro and in plants.

  9. Off-target Effects in CRISPR/Cas9-mediated Genome Engineering

    PubMed Central

    Zhang, Xiao-Hui; Tee, Louis Y; Wang, Xiao-Gang; Huang, Qun-Shan; Yang, Shi-Hua

    2015-01-01

    CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)—RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site—is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype–phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology. PMID:26575098

  10. CRISPR/Cas9-Mediated Genome Editing of Epigenetic Factors for Cancer Therapy.

    PubMed

    Yao, Shaohua; He, Zhiyao; Chen, Chong

    2015-07-01

    Advances in engineered recombinant nuclease have provided facile and reliable methods for genome editing. Especially with the development of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein-9 nuclease) system, the discovery of various versions of Cas9 proteins and delivery carriers, it is now practicable to introduce desired mutations into the genome, to correct disease-related mutations, and to activate or suppress genes of interest. Epigenetic regulators are often disturbed in cancer cells and are essential for the transformation of normal to cancerous cells. Tumor-related epigenetic alterations or epigenetic factor mutations play a major part during the various steps of carcinogenesis and affect a variety of cancer-related genes and a wide range of cancerous phenotypes. Therefore, epigenetic regulatory enzymes might be candidate targets for cancer therapy. In this review, we discuss prospects of CRISPR/Cas9-based genome editing in targeting epigenetics for cancer gene therapy.

  11. Controlling UCAVs by JTACs in CAS missions

    NASA Astrophysics Data System (ADS)

    Kumaş, A. E.

    2014-06-01

    By means of evolving technology, capabilities of UAVs (Unmanned Aerial Vehicle)s are increasing rapidly. This development provides UAVs to be used in many different areas. One of these areas is CAS (Close Air Support) mission. UAVs have several advantages compared to manned aircraft, however there are also some problematic areas. The remote controlling of these vehicles from thousands of nautical miles away via satellite may lead to various problems both ethical and tactical aspects. Therefore, CAS missions require a good level of ALI (Air-Land Integration), a high SA (situational awareness) and precision engagement. In fact, there is an aware friendly element in the target area in CAS missions, unlike the other UAV operations. This element is an Airman called JTAC (Joint Terminal Attack Controller). Unlike the JTAC, UAV operators are too far away from target area and use the limited FOV (Field of View) provided by camera and some other sensor data. In this study, target area situational awareness of a UAV operator and a JTAC, in a high-risk mission for friendly ground forces and civilians such as CAS, are compared. As a result of this comparison, answer to the question who should control the UCAV (Unmanned Combat Aerial Vehicle) in which circumstances is sought. A literature review is made in UAV and CAS fields and recent air operations are examined. The control of UCAV by the JTAC is assessed by SWOT analysis and as a result it is deduced that both control methods can be used in different situations within the framework of the ROE (Rules Of Engagement) is reached.

  12. Designing flexible instructional space for teaching introductory physics with emphasis on inquiry and collaborative active learning

    NASA Astrophysics Data System (ADS)

    Bykov, Tikhon

    2010-03-01

    In recent years McMurry University's introductory physics curriculum has gone through a series of significant changes to achieve better integration of traditional course components (lecture/lab/discussion) by means of instructional design and technology. A system of flexible curriculum modules with emphasis on inquiry-based teaching and collaborative active learning has been introduced. To unify module elements, a technology suite has been used that consists of Tablet PC's and software applications including Physlets, tablet-adapted personal response system, PASCO data acquisition systems, and MS One-note collaborative writing software. Adoption of the new teaching model resulted in reevaluation of existing instructional spaces. The new teaching space will be created during the renovation of the McMurry Science Building. This space will allow for easy transitions between lecture and laboratory modes. Movable partitions will be used to accommodate student groups of different sizes. The space will be supportive of small peer-group activities with easy-to-reconfigure furniture, multiple white and black board surfaces and multiple projection screens. The new space will be highly flexible to account for different teaching functions, different teaching modes and learning styles.

  13. Fundamental Physics Activities in the Hme Directorate of the European Space Agency

    NASA Astrophysics Data System (ADS)

    Cacciapuoti, Luigi; Minster, Olivier

    The Human Spaceflight, Microgravity, and Exploration (HME) Directorate of the European Space Agency is strongly involved in fundamental physics research. One of the major activities in this field is represented by the ACES (Atomic Clock Ensemble in Space) mission. ACES will demonstrate the high performances of a new generation of atomic clocks in the microgravity environment of the International Space Station (ISS). Following ACES, a vigorous research program has been recently approved to develop a second generation of atomic quantum sensors for space applications: atomic clocks in the optical domain, aiming at fractional frequency stability and accuracy in the low 10-18 regime; inertial sensors based on matter-wave interferometry for the detection of tiny accelerations and rotations; a facility to study degenerate Bose gases in space. Tests of quantum physics on large distance scales represent another important issue addressed in the HME program. A quantum communication optical terminal has been proposed to perform a test of Bell's inequalities on pairs of entangled photons emitted by a source located on the ISS and detected by two ground stations. In this paper, present activities and future plans will be described and discussed.

  14. Aeronautics and Space Report of the President, Fiscal Year 2002 Activities

    NASA Technical Reports Server (NTRS)

    2002-01-01

    Fiscal Year (FY) 2002 brought advances on many fronts in support of NASAs new vision, announced by Administrator Sean OKeefe on April 12, to improve life here, to extend life to there, to find life beyond. NASA successfully carried out four Space Shuttle missions, including three to the International Space Station (ISS) and one servicing mission to the Hubble Space Telescope (HST). By the end of the fiscal year, humans had occupied the ISS continuously for 2 years. NASA also managed five expendable launch vehicle (ELV) missions and participated in eight international cooperative ELV launches. In the area of space science, two of the Great Observatories, the Hubble Space Telescope and the Chandra X-Ray Observatory, continued to make spectacular observations. The Mars Global Surveyor and Mars Odyssey carried out their mapping missions of the red planet in unprecedented detail. Among other achievements, the Near Earth Asteroid Rendezvous (NEAR) Shoemaker spacecraft made the first soft landing on an asteroid, and the Solar and Heliospheric Observatory (SOHO) monitored a variety of solar activity, including the largest sunspot observed in 10 years. The education and public outreach program stemming from NASAs space science missions continues to grow. In the area of Earth science, attention focused on completing the first Earth Observing Satellite series. Four spacecraft were successfully launched. The goal is to understand our home planet as a system, as well as how the global environment responds to change.

  15. In vivo genome editing using Staphylococcus aureus Cas9

    PubMed Central

    Ran, F. Ann; Cong, Le; Yan, Winston X.; Scott, David A.; Gootenberg, Jonathan S.; Kriz, Andrea J.; Zetsche, Bernd; Shalem, Ophir; Wu, Xuebing; Makarova, Kira S.; Koonin, Eugene; Sharp, Phillip A.; Zhang, Feng

    2015-01-01

    The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that employ the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologs and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being >1kb shorter. We packaged SaCas9 and its sgRNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further demonstrate the power of using BLESS to assess the genome-wide targeting specificity of SaCas9 and SpCas9, and show that SaCas9 can mediate genome editing in vivo with high specificity. PMID:25830891

  16. The CRISPR/Cas9 system for gene editing and its potential application in pain research

    PubMed Central

    Sun, Linlin; Lutz, Brianna Marie; Tao, Yuan-Xiang

    2016-01-01

    The CRISPR/Cas9 system is a research hotspot in genome editing and regulation. Currently, it is used in genomic silencing and knock-in experiments as well as transcriptional activation and repression. This versatile system consists of two components: a guide RNA (gRNA) and a Cas9 nuclease. Recognition of a genomic DNA target is mediated through base pairing with a 20-base gRNA. The latter further recruits the Cas9 endonuclease protein to the target site and creates double-stranded breaks in the target DNA. Compared with traditional genome editing directed by DNA-binding protein domains, this short RNA-directed Cas9 endonuclease system is simple and easily programmable. Although this system may have off-target effects and in vivo delivery and immune challenges, researchers have employed this system in vivo to establish disease models, study specific gene functions under certain disease conditions, and correct genomic information for disease treatment. In regards to pain research, the CRISPR/Cas9 system may act as a novel tool in gene correction therapy for pain-associated hereditary diseases and may be a new approach for RNA-guided transcriptional activation or repression of pain-related genes. In addition, this system is also applied to loss-of-function mutations in pain-related genes and knockin of reporter genes or loxP tags at pain-related genomic loci. The CRISPR/Cas9 system will likely be carried out widely in both bench work and clinical settings in the pain field. PMID:27500183

  17. Office of Commercial Programs' research activities for Space Station Freedom utilization

    NASA Astrophysics Data System (ADS)

    Fountain, James A.

    One of the objectives of the Office of Commercial Programs (OCP) is to encourage, enable, and help implement space research which meets the needs of the U.S. industrial sector. This is done mainly through seventeen Centers for the Commercial Development of Space (CCDS's) which are located throughout the United States. The CCDS's are composed of members from U.S. companies, universities, and other government agencies. These Centers are presently engaged in industrial research in space using a variety of carriers to reach low Earth orbit. One of the goals is to produce a body of experience and knowledge that will allow U.S. industrial entities to make informed decisions regarding their participation in commercial space endeavors. A total of 32 items of payload hardware were built to date. These payloads have flown in space a total of 73 times. The carriers range from the KC-135 parabolic aircraft and expendable launch vehicles to the Space Shuttle. This range of carriers allows the experimenter to evolve payloads in complexity and cost by progressively extending the time in microgravity. They can start with a few seconds in the parabolic aircraft and go to several minutes on the rocket flights, before they progress to the complexities of manned flight on the Shuttle. Next year, two new capabilities will become available: COMET, an expendable-vehicle-launched experiment capsule that can carry experiments aloft for thirty days; and SPACEHAB, a new Shuttle borne module which will greatly add to the capability to accommodate small payloads. All of these commercial research activities and carrier capabilities are preparing the OCP to evolve those experiments that prove successful to Space Station Freedom. OCP and the CCDS's are actively involved in Space Station design and utilization planning and have proposed a set of experiments to be launched in 1996 and 1997. These experiments are to be conducted both internal and external to Space Station Freedom and will

  18. Office of Commercial Programs' research activities for Space Station Freedom utilization

    NASA Technical Reports Server (NTRS)

    Fountain, James A.

    1992-01-01

    One of the objectives of the Office of Commercial Programs (OCP) is to encourage, enable, and help implement space research which meets the needs of the U.S. industrial sector. This is done mainly through seventeen Centers for the Commercial Development of Space (CCDS's) which are located throughout the United States. The CCDS's are composed of members from U.S. companies, universities, and other government agencies. These Centers are presently engaged in industrial research in space using a variety of carriers to reach low Earth orbit. One of the goals is to produce a body of experience and knowledge that will allow U.S. industrial entities to make informed decisions regarding their participation in commercial space endeavors. A total of 32 items of payload hardware were built to date. These payloads have flown in space a total of 73 times. The carriers range from the KC-135 parabolic aircraft and expendable launch vehicles to the Space Shuttle. This range of carriers allows the experimenter to evolve payloads in complexity and cost by progressively extending the time in microgravity. They can start with a few seconds in the parabolic aircraft and go to several minutes on the rocket flights, before they progress to the complexities of manned flight on the Shuttle. Next year, two new capabilities will become available: COMET, an expendable-vehicle-launched experiment capsule that can carry experiments aloft for thirty days; and SPACEHAB, a new Shuttle borne module which will greatly add to the capability to accommodate small payloads. All of these commercial research activities and carrier capabilities are preparing the OCP to evolve those experiments that prove successful to Space Station Freedom. OCP and the CCDS's are actively involved in Space Station design and utilization planning and have proposed a set of experiments to be launched in 1996 and 1997. These experiments are to be conducted both internal and external to Space Station Freedom and will

  19. 32 CFR Appendix C to Part 169a - Simplified Cost Comparison and Direct Conversion of CAs

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... cost comparisons. 3. For activities large in total size (including those with a mix of civilian and... 32 National Defense 1 2014-07-01 2014-07-01 false Simplified Cost Comparison and Direct Conversion... 169a—Simplified Cost Comparison and Direct Conversion of CAs A. This appendix provides guidance...

  20. 32 CFR Appendix C to Part 169a - Simplified Cost Comparison and Direct Conversion of CAs

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... cost comparisons. 3. For activities large in total size (including those with a mix of civilian and... 32 National Defense 1 2011-07-01 2011-07-01 false Simplified Cost Comparison and Direct Conversion... 169a—Simplified Cost Comparison and Direct Conversion of CAs A. This appendix provides guidance...