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Sample records for action study cas

  1. actions immunoallergiques graves aux antibacillaires: à propos de 10 cas

    PubMed Central

    Alami, Sabah El Machichi; Hammi, Sanae; Bourkadi, Jamal Eddine

    2014-01-01

    L'hypersensibilité aux antituberculeux est l'un des effets secondaires imprévisibles qui apparait chez 4 à 5 % de la population exposée et s’élève à 25% chez les sujets VIH positifs. Dans notre étude parmi 39 patients ayant présenté des réactions immunoallergiques, 10 avaient des formes graves. Le délai moyen d'apparition des signes était de 23 jours. Les réactions immunoallergiques observées étaient 5 cas de toxidermie généralisée fébrile, un cas de Dress syndrome, un cas de neutropénie, un cas de pancitopénie et 2 cas de thrombopénie. Tous nos patients avaient bien évolué cliniquement et bactériologiquement après l'adoption d'un régime thérapeutique excluant le ou les médicaments incriminés. En pratique, si l'effet indésirable imputé à un antituberculeux est grave, il est impératif de l'arrêter, de traiter l'incident et d'associer une autre molécule chez certains cas. Notre étude a montré une fréquence significative des complications graves probablement sous-estimée, surtout dans les pays fortement touchés par l'infection HIV.

  2. The Assessment of a Tutoring Program to Meet CAS Standards Using a SWOT Analysis and Action Plan

    ERIC Educational Resources Information Center

    Fullmer, Patricia

    2009-01-01

    This article summarizes the use of SWOT (Strengths, Weaknesses, Opportunities, and Threats) analysis and subsequent action planning as a tool of self-assessment to meet CAS (Council for the Advancement of Standards in Higher Education) requirements for systematic assessment. The use of the evaluation results to devise improvements to increase the…

  3. Interactive Intergenerational Learning in the Context of CAS in the IB Diploma Programme: A UK Case Study

    ERIC Educational Resources Information Center

    Cambridge, James; Simandiraki, Anna

    2006-01-01

    Creativity, Action, Service (CAS) forms a core component of the International Baccalaureate Diploma Programme. CAS frequently includes intergenerational practice involving school students and older adults. This article reports the development of a typology to describe and analyse learning that arises from intergenerational practice in three…

  4. NASA Controller Acceptability Study 1(CAS-1) Experiment Description and Initial Observations

    NASA Technical Reports Server (NTRS)

    Chamberlain, James P.; Consiglio, Maria C.; Comstock, James R., Jr.; Ghatas, Rania W.; Munoz, Cesar

    2015-01-01

    This paper describes the Controller Acceptability Study 1 (CAS-1) experiment that was conducted by NASA Langley Research Center personnel from January through March 2014 and presents partial CAS-1 results. CAS-1 employed 14 air traffic controller volunteers as research subjects to assess the viability of simulated future unmanned aircraft systems (UAS) operating alongside manned aircraft in moderate-density, moderate-complexity Class E airspace. These simulated UAS were equipped with a prototype pilot-in-the-loop (PITL) Detect and Avoid (DAA) system, specifically the Self-Separation (SS) function of such a system based on Stratway+ software to replace the see-and-avoid capabilities of manned aircraft pilots. A quantitative CAS-1 objective was to determine horizontal miss distance (HMD) values for SS encounters that were most acceptable to air traffic controllers, specifically HMD values that were assessed as neither unsafely small nor disruptively large. HMD values between 0.5 and 3.0 nautical miles (nmi) were assessed for a wide array of encounter geometries between UAS and manned aircraft. The paper includes brief introductory material about DAA systems and their SS functions, followed by descriptions of the CAS-1 simulation environment, prototype PITL SS capability, and experiment design, and concludes with presentation and discussion of partial CAS-1 data and results.

  5. Spectroscopic studies of the small-amplitude Cepheid SU Cas

    NASA Astrophysics Data System (ADS)

    Usenko, I. A.; Klochkova, V. G.; Tavolzhanskaya, N. S.

    2013-09-01

    A new set of 16 high-resolution spectra for the small-amplitude Cepheid SU Cas obtained in 2007-2009 has allowed us to determine its atmospheric parameters ( T eff = 6345 ± 30 K, log g = 2.40, V t = 3.25 km s-1) and to measure its radial velocities. The latter were added to the general list of radial velocities (375 estimates) obtained in the last 90 years. Using a frequency analysis, we have refined the pulsation and orbital periods of the Cepheid. Apart from the well-known fundamental pulsation period [Figure not available: see fulltext.], we have detected a possible secondary period of [Figure not available: see fulltext.]. Their ratio of 0.96 suggests the existence of nonradial pulsations in the Cepheid's atmosphere. Based on photoelectric photometry in the last 60 years, we have shown that the effective temperature undergoes cyclic secular changes of ±200 K with an unknown period. The mean effective temperature T eff = 6395 ± 52 K estimated from photometric data agrees well with our estimate from spectroscopic data. The variations of the mean color index, effective temperature, and γ-velocity (in 90 years of observations) point to a possible orbital motion of the well-known hot companion with the most probable periods of [Figure not available: see fulltext.], [Figure not available: see fulltext.], and [Figure not available: see fulltext.]. The elemental abundances in the atmosphere of SU Cas confirm the conclusion that this Cepheid is a typical yellow supergiant after the first dredge-up. Our T eff estimate gives a radius of 32 R ⊙ and a distance of 455 pc for it, which is inconsistent with its membership in the open cluster Alessi 95. The question about the pulsation mode of SU Cas still remains open.

  6. Spectroscopic studies of three Cepheids with high positive pulsation period increments: SZ Cas, BY Cas, and RU Sct

    NASA Astrophysics Data System (ADS)

    Usenko, I. A.; Klochkova, V. G.

    2015-07-01

    Three high-resolution spectra have been taken at different times with the 6-m SAO RAS telescope (LYNX and PFES spectrographs) for three Cepheids exhibiting high positive period increments: the small-amplitude (DCEPS) SZ Cas and BY Cas and the classical (DCEP) RU Sct. SZ Cas and RU Sct are members of the Galactic open clusters χ and h Per and Trump 35, respectively. Analysis of the spectra has shown that the interstellar Na I D1 and D2 lines in all objects are considerably stronger than the atmospheric ones and are redshifted in SZ Cas and BY Cas and blushifted in RU Sct. The core of the H α absorption line in BY Cas has an asymmetric knifelike shape, while RU Sct exhibits an intense emission in the blue wing of this line. Such phenomena are observed in long-period Cepheids and bright hypergiants with an extended envelope. In this case, the strong Mg Ib 5183.62 Å and Ba II 5853.67, 6141.713, and 6496.90 Å lines with low χlow in SZ Cas and RU Sct also show characteristic knifelike profiles with an asymmetry in the red region, while the Ba II 4934.095 Å line shows similar profiles in the blue one. The absorption lines of neutral atoms and singly ionized metals with different lowerlevel excitation potentials exhibit different degrees of asymmetry: from a pronounced one with secondary components in BY Cas (similar to those in the small-amplitude Cepheid BG Cru pulsating in the first overtone and having an envelope) to its insignificance or virtual absence in SZ Cas and RU Sct. Analysis of the secular changes in mean T eff determined from photometric color indices and spectra over the last 55 years for these stars has revealed periodic fluctuations of 200 K for SZ Cas and BY Cas and 500 K for RU Sct. For SZ Cas and RU Sct, T eff determined in some years from some color indices show much lower values, which together with the temperature fluctuations can be associated with mass loss and dust formation. Based on these facts, we hypothesize the existence of

  7. 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. PMID:26950513

  8. CRISPR/Cas9 as Tool for Functional Study of Genes Involved in Preimplantation Embryo Development

    PubMed Central

    Kwon, Jeongwoo; Namgoong, Suk; Kim, Nam-Hyung

    2015-01-01

    The CRISPR/Cas9 system has proven to be an efficient gene-editing tool for genome modification of cells and organisms. However, the applicability and efficiency of this system in pig embryos have not been studied in depth. Here, we aimed to remove porcine OCT4 function as a model case using the CRISPR/Cas9 system. Injection of Cas9 and single-guide RNA (sgRNA) against OCT4 decreased the percentages of OCT4-positive embryos to 37–50% of total embryos, while ~100% of control embryos exhibited clear OCT4 immunostaining. We assessed the mutation status near the guide sequence using polymerase chain reaction (PCR) and DNA sequencing, and a portion of blastocysts (20% in exon 2 and 50% in exon 5) had insertions/deletions near protospacer-adjacent motifs (PAMs). Different target sites had frequent deletions, but different concentrations of sgRNA made no impact. OCT4 mRNA levels dramatically decreased at the 8-cell stage, and they were barely detectable in blastocysts, while mRNA levels of other genes, including NANOG, and CDX2 were not affected. In addition, the combination of two sgRNAs led to large-scale deletion (about 1.8 kb) in the same chromosome. Next, we injected an enhanced green fluorescent protein (eGFP) vector targeting the OCT4 exon with Cas9 and sgRNA to create a knockin. We confirmed eGFP fluorescence in blastocysts in the inner cell mass, and also checked the mutation status using PCR and DNA sequencing. A significant portion of blastocysts had eGFP sequence insertions near PAM sites. The CRISPR/CAS9 system provides a good tool for gene functional studies by deleting target genes in the pig. PMID:25775469

  9. CLIM-RUN: Tourism cas study over the French Alps

    NASA Astrophysics Data System (ADS)

    Dubois, C.

    2012-04-01

    Climate information for societal use has becoming a major challenge for tourism management and adaptation in a context of strong climate variability and change. Within the CLIMRUN EU FP7, a case study on summer tourism in the French Alps has been identify. I will introduce the bottom-up approach use in the project where stakeholders and local users meet with climate experts. From those meetings, they thus identify the climate dependence and information which impact their summer activities over this region. All the activities are located in a mountainous region where outdoor leisure is the main economic driver of the region. It has emerged that the climate requirements are as well on past as on future climate information. On one side, the past climate parameters are found to be an invaluable information to evaluate the climate dependence of the different activities. A better knowledge as well as a growing interest in climate variability has been express to quantify the climate dependence on their activities. On the other side, the future climate information requested mainly on seasonal to decadal timescale. A particular interest has been express on the snow cover at the end of the winter season, evolution of heavy precipitations, heatwave, air temperatures and well as the water temperature of the mountainous lakes. Those climate variables are used to create comfort index under climate change. All those targeted climate information are based on on-going projects as well as future model development.

  10. Final corrective action study for the former CCC/USDA facility in Ramona, Kansas.

    SciTech Connect

    LaFreniere, L. M.

    2011-04-20

    Past operations at a grain storage facility formerly leased and operated by the Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA) in Ramona, Kansas, resulted in low concentrations of carbon tetrachloride in groundwater that slightly exceed the regulatory standard in only one location. As requested by the Kansas Department of Health and Environment, the CCC/USDA has prepared a Corrective Action Study (CAS) for the facility. The CAS examines corrective actions to address groundwater impacted by the former CCC/USDA facility but not releases caused by other potential groundwater contamination sources in Ramona. Four remedial alternatives were considered in the CAS. The recommended remedial alternative in the CAS consists of Environmental Use Control to prevent the inadvertent use of groundwater as a water supply source, coupled with groundwater monitoring to verify the continued natural improvement in groundwater quality. The Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA) has directed Argonne National Laboratory to prepare a Corrective Action Study (CAS), consistent with guidance from the Kansas Department of Health and Environment (KDHE 2001a), for the CCC/USDA grain storage facility formerly located in Ramona, Kansas. This effort is pursuant to a KDHE (2007a) request. Although carbon tetrachloride levels at the Ramona site are low, they remain above the Kansas Tier 2 risk-based screening level (RBSL) and the U.S. Environmental Protection Agency (EPA) maximum contaminant level (MCL) of 5 {micro}g/L (Kansas 2003, 2004). In its request for the CAS, the KDHE (2007a) stated that, because of these levels, risk is associated with potential future exposure to contaminated groundwater. The KDHE therefore determined that additional measures are warranted to limit future use of the property and/or exposure to contaminated media as part of site closure. The KDHE further requested comparison of at least two corrective

  11. CRISPR/Cas9 Promotes Functional Study of Testis Specific X-Linked Gene In Vivo

    PubMed Central

    Jiang, Xue; Chen, Yuxi; Zhang, Zhen; Zhang, Xiya; Liang, Puping; Zhan, Shaoquan; Cao, Shanbo; Songyang, Zhou; Huang, Junjiu

    2015-01-01

    Mammalian spermatogenesis is a highly regulated multistage process of sperm generation. It is hard to uncover the real function of a testis specific gene in vitro since the in vitro model is not yet mature. With the development of the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9) system, we can now rapidly generate knockout mouse models of testis specific genes to study the process of spermatogenesis in vivo. SYCP3-like X-linked 2 (SLX2) is a germ cell specific component, which contains a Cor1 domain and belongs to the XLR (X-linked, lymphocyte regulated) family. Previous studies suggested that SLX2 might play an important role in mouse spermatogenesis based on its subcellular localization and interacting proteins. However, the function of SLX2 in vivo is still elusive. Here, to investigate the functions of SLX2 in spermatogenesis, we disrupted the Slx2 gene by using the CRISPR/Cas9 system. Since Slx2 is a testis specific X-linked gene, we obtained knockout male mice in the first generation and accelerated the study process. Compared with wild-type mice, Slx2 knockout mice have normal testis and epididymis. Histological observation of testes sections showed that Slx2 knockout affected none of the three main stages of spermatogenesis: mitosis, meiosis and spermiogenesis. In addition, we further confirmed that disruption of Slx2 did not affect the number of spermatogonial stem cells, meiosis progression or XY body formation by immunofluorescence analysis. As spermatogenesis was normal in Slx2 knockout mice, these mice were fertile. Taken together, we showed that Slx2 itself is not an essential gene for mouse spermatogenesis and CRISPR/Cas9 technique could speed up the functional study of testis specific X-linked gene in vivo. PMID:26599493

  12. CRISPR/Cas9 genome editing throws descriptive 3-D genome folding studies for a loop.

    PubMed

    Beagan, Jonathan A; Phillips-Cremins, Jennifer E

    2016-07-01

    CRISPR/Cas9 genome editing studies have recently shed new light into the causal link between the linear DNA sequence and 3-D chromatin architecture. Here we describe current models for the folding of genomes into a nested hierarchy of higher-order structures and discuss new insights into the organizing principles governing genome folding at each length scale. WIREs Syst Biol Med 2016, 8:286-299. doi: 10.1002/wsbm.1338 For further resources related to this article, please visit the WIREs website. PMID:27265842

  13. A Class Exercise: Studying the Eclipsing Binary Star RZ Cas Through Visual Observations

    NASA Astrophysics Data System (ADS)

    Balonek, T. J.; Davis, S. M.

    2000-05-01

    As part of the sophomore-junior level "Astronomical Techniques" course at Colgate University, students learn just how much science they can do with simple tools: a pair of binoculars, a clock, and pencil and paper. The students study the Algol type visual eclipsing binary star system RZ Cassiopeiae: observing and making a light curve for the primary minimum, determining the time of minimum using several techniques, calculating the binary star system's orbital period, and determining changes in the system's period over a thirty year interval by constructing an O-C curve. Through a series of preparatory exercises, the students learn how to read star maps and use the unaided eye, binoculars and telescopes to locate star fields and make visual magnitude measurements. By making multiple measurements of stars in the field of RZ Cas on several nights, the students determine the accuracy they can achieve in estimating the visual magnitude of a star -- typically 0.2 magnitude. (Some students even accidentally discover that one of the stars in the field is a variable star!) With this experience, the students use binoculars to observe the four hour primary eclipse of RZ Cas (magnitude 6.2 - 7.7), making magnitude measurements every five minutes. A light curve is then plotted. Several methods are used to determine the time of minimum, which is then converted to heliocentric Julian day. Using times of minima determined by former students (and the instructor) in previous years dating from 1968 to the present, the students determine the average period to a tenth of a second second. By constructing an O-C curve from the class's data and that obtained by the AAVSO, changes in the period of RZ Cas are noticeable -- possibly due to mass transfer in the system. It will be interesting for future classes to build on this knowledge using the primitive tools of our not so distant past.

  14. 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…

  15. Mouse genome engineering using CRISPR-Cas9 for study of immune function

    PubMed Central

    Pelletier, Stephane; Gingras, Sebastien; Green, Douglas R.

    2016-01-01

    Clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated (Cas9) technology has proven a formidable addition to our armory of approaches for genomic editing. Derived from pathways in archaea and bacteria that mediate the resistance to exogenous genomic material, the CRISPR-Cas9 system utilizes a short single guide RNA (sgRNA) to direct the endonuclease Cas9 to virtually anywhere in the genome. Upon targeting, Cas9 generates DNA double strand breaks (DSBs) and facilitates the repair or insertion of mutations, insertion of recombinase recognition sites or large DNA elements. Here, we discuss the practical advantages of the CRISPR-Cas9 system over conventional and other nuclease-based targeting technologies and provide suggestions for the use of this technology to address immunological questions. PMID:25607456

  16. Using CRISPR/Cas to study gene function and model disease in vivo.

    PubMed

    Tschaharganeh, Darjus F; Lowe, Scott W; Garippa, Ralph J; Livshits, Geulah

    2016-09-01

    The recent discovery of the CRISPR/Cas system and repurposing of this technology to edit a variety of different genomes have revolutionized an array of scientific fields, from genetics and translational research, to agriculture and bioproduction. In particular, the prospect of rapid and precise genome editing in laboratory animals by CRISPR/Cas has generated an immense interest in the scientific community. Here we review current in vivo applications of CRISPR/Cas and how this technology can improve our knowledge of gene function and our understanding of biological processes in animal models. PMID:27149548

  17. Excited-State Photolytic Mechanism of Cyclopentene Containing a Group 14 Element: An MP2-CAS//CASSCF Study.

    PubMed

    Su, Ming-Der

    2015-08-13

    The potential energy surfaces corresponding to the photolytic reactions of 1,2-dimethyl-cyclopentene, 3,4-dimethyl-silacyclopent-3-ene, and 3,4-dimethyl-germacyclopent-3-ene were investigated by employing the CAS(6,6)/6-311G(d) and MP2-CAS-(6,6)/6-311++G(3df,3pd)//CAS(6,6)/6-311G(d) methods. Also, six kinds of substituted germacyclopent-3-ene were used as model reactants by way of the CASSCF and MP2-CAS methods to study their photolytic mechanisms. The theoretical findings indicate that the photolysis of the above reactants all adopt the same reaction path as follows: reactant → Franck-Condon region → conical intersection → germylene and 1,3-butadiene. However, the theoretical results demonstrate that no photolysis ((1)(π →π*)) can be observed in the 1,2-dimethyl-cyclopentene system. Above all, the theoretical investigations strongly suggest that both steric effects, originating from the bulky substituents, and the atomic radius of the group 14 element (C, Si, and Ge) play a crucial role in determining the cis/trans selectivity of the conformation of 1,3-butadiene during their photolytic reactions. PMID:26204240

  18. Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells.

    PubMed

    Moorthy, Sakthi D; Mitchell, Jennifer A

    2016-01-01

    Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus(129) x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells. PMID:27078492

  19. mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence

    PubMed Central

    Finnigan, Gregory C.; Thorner, Jeremy

    2016-01-01

    Genome editing exploiting CRISPR/Cas9 has been adopted widely in academia and in the biotechnology industry to manipulate DNA sequences in diverse organisms. Molecular engineering of Cas9 itself and its guide RNA, and the strategies for using them, have increased efficiency, optimized specificity, reduced inappropriate off-target effects, and introduced modifications for performing other functions (transcriptional regulation, high-resolution imaging, protein recruitment, and high-throughput screening). Moreover, Cas9 has the ability to multiplex, i.e., to act at different genomic targets within the same nucleus. Currently, however, introducing concurrent changes at multiple loci involves: (i) identification of appropriate genomic sites, especially the availability of suitable PAM sequences; (ii) the design, construction, and expression of multiple sgRNA directed against those sites; (iii) potential difficulties in altering essential genes; and (iv) lingering concerns about “off-target” effects. We have devised a new approach that circumvents these drawbacks, as we demonstrate here using the yeast Saccharomyces cerevisiae. First, any gene(s) of interest are flanked upstream and downstream with a single unique target sequence that does not normally exist in the genome. Thereafter, expression of one sgRNA and cotransformation with appropriate PCR fragments permits concomitant Cas9-mediated alteration of multiple genes (both essential and nonessential). The system we developed also allows for maintenance of the integrated, inducible Cas9-expression cassette or its simultaneous scarless excision. Our scheme—dubbed mCAL for “Multiplexing of Cas9 at Artificial Loci”—can be applied to any organism in which the CRISPR/Cas9 methodology is currently being utilized. In principle, it can be applied to install synthetic sequences into the genome, to generate genomic libraries, and to program strains or cell lines so that they can be conveniently (and repeatedly

  20. 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

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

    PubMed

    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

  2. mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence.

    PubMed

    Finnigan, Gregory C; Thorner, Jeremy

    2016-01-01

    Genome editing exploiting CRISPR/Cas9 has been adopted widely in academia and in the biotechnology industry to manipulate DNA sequences in diverse organisms. Molecular engineering of Cas9 itself and its guide RNA, and the strategies for using them, have increased efficiency, optimized specificity, reduced inappropriate off-target effects, and introduced modifications for performing other functions (transcriptional regulation, high-resolution imaging, protein recruitment, and high-throughput screening). Moreover, Cas9 has the ability to multiplex, i.e., to act at different genomic targets within the same nucleus. Currently, however, introducing concurrent changes at multiple loci involves: (i) identification of appropriate genomic sites, especially the availability of suitable PAM sequences; (ii) the design, construction, and expression of multiple sgRNA directed against those sites; (iii) potential difficulties in altering essential genes; and (iv) lingering concerns about "off-target" effects. We have devised a new approach that circumvents these drawbacks, as we demonstrate here using the yeast Saccharomyces cerevisiae First, any gene(s) of interest are flanked upstream and downstream with a single unique target sequence that does not normally exist in the genome. Thereafter, expression of one sgRNA and cotransformation with appropriate PCR fragments permits concomitant Cas9-mediated alteration of multiple genes (both essential and nonessential). The system we developed also allows for maintenance of the integrated, inducible Cas9-expression cassette or its simultaneous scarless excision. Our scheme-dubbed mCAL for " M: ultiplexing of C: as9 at A: rtificial L: oci"-can be applied to any organism in which the CRISPR/Cas9 methodology is currently being utilized. In principle, it can be applied to install synthetic sequences into the genome, to generate genomic libraries, and to program strains or cell lines so that they can be conveniently (and repeatedly

  3. Corrective Action Investigation Plan for Corrective Action Unit 550: Smoky Contamination Area Nevada National Security Site, Nevada, Revision 0

    SciTech Connect

    Grant Evenson

    2012-05-01

    Corrective Action Unit (CAU) 550 is located in Areas 7, 8, and 10 of the Nevada National Security Site, which is approximately 65 miles northwest of Las Vegas, Nevada. CAU 550, Smoky Contamination Area, comprises 19 corrective action sites (CASs). Based on process knowledge of the releases associated with the nuclear tests and radiological survey information about the location and shape of the resulting contamination plumes, it was determined that some of the CAS releases are co-located and will be investigated as study groups. This document describes the planned investigation of the following CASs (by study group): (1) Study Group 1, Atmospheric Test - CAS 08-23-04, Atmospheric Test Site T-2C; (2) Study Group 2, Safety Experiments - CAS 08-23-03, Atmospheric Test Site T-8B - CAS 08-23-06, Atmospheric Test Site T-8A - CAS 08-23-07, Atmospheric Test Site T-8C; (3) Study Group 3, Washes - Potential stormwater migration of contaminants from CASs; (4) Study Group 4, Debris - CAS 08-01-01, Storage Tank - CAS 08-22-05, Drum - CAS 08-22-07, Drum - CAS 08-22-08, Drums (3) - CAS 08-22-09, Drum - CAS 08-24-03, Battery - CAS 08-24-04, Battery - CAS 08-24-07, Batteries (3) - CAS 08-24-08, Batteries (3) - CAS 08-26-01, Lead Bricks (200) - CAS 10-22-17, Buckets (3) - CAS 10-22-18, Gas Block/Drum - CAS 10-22-19, Drum; Stains - CAS 10-22-20, Drum - CAS 10-24-10, Battery. These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives (CAAs). Additional information will be obtained by conducting a corrective action investigation before evaluating CAAs and selecting the appropriate corrective action for each study group. The results of the field investigation will support a defensible evaluation of viable CAAs that will be presented in the Corrective Action Decision Document. The sites will be investigated based on the data quality objectives (DQOs) developed

  4. Following the Action in Action Learning: Towards Ethnomethodological Studies of (Critical) Action Learning

    ERIC Educational Resources Information Center

    Fox, Steve

    2009-01-01

    Action learning is a pedagogical practice that helps participants learn by talking about their workplace action with fellow participants ("comrades in adversity") in their action learning set. This paper raises questions about the action in action learning, such as: how do members of an action learning set learn from and through each other? How do…

  5. A laser study of the blue electronic transitions of CaS

    NASA Technical Reports Server (NTRS)

    Jarman, C. N.; Hailey, R. A.; Bernath, P. F.

    1992-01-01

    Three electronic transitions of CaS are observed in the blue region of the spectrum. The vibrational bands in these transitions span thousands per cm owing to a large change in geometry. Approximately 1000/cm portion of the spectrum containing these bands is recorded at high resolution by dye laser excitation spectroscopy and analyzed. The states all have Omega = 1, and are tentatively labeled G 1Pi, F 1Pi, and f 3Pi 1. Spectroscopic constants are generated for all three upper states and for the X 1Sigma(+) state from a fit to 4340 individual lines. These constants are used to generate potential curves and Franck-Condon factors.

  6. Action Learning in ActionAid Nepal: A Case Study

    ERIC Educational Resources Information Center

    Lustig, Patricia; Rai, Deep Ranjani

    2009-01-01

    This article describes an example of how action learning was used as a framework for an organisational intervention to fundamentally change the organisational culture over a period of time. It also identifies our learning over that period of time and what worked well (and not so well) in an International Non-Governmental Organisation in Nepal.

  7. Validation of Subject Areas of CAS Professional Studies Standards for Master's Level Student Affairs Professional Preparation Programs

    ERIC Educational Resources Information Center

    Young, Dallin George; Dean, Laura A.

    2015-01-01

    The standards for Master's Level Student Affairs Professional Preparation Programs, first published in 1986, were among the first standards published by the Council for the Advancement of Standards in Higher Education (CAS; Ebbers & Kruempel, 1992). With the latest revision in 2012, the CAS standards for preparation of student affairs…

  8. Co-Alignment System (CAS) study. Report on task 1-3. [Solar Extreme Ultraviolet Telescope and Spectrometer pointing system

    NASA Technical Reports Server (NTRS)

    Anderson, N. T.

    1980-01-01

    The design of a suitable coalignment system (CAS) for the Solar Extreme Ultraviolet Telescope and Spectrometer (SEUTS) is presented. The CAS provides offset adjustment capabilities to SEUTS which will be mounted on a single large pointing system with other devices. The suitability of existing designs is determined and modifications are suggested.

  9. Understanding communicative actions: a repetitive TMS study.

    PubMed

    Stolk, Arjen; Noordzij, Matthijs L; Volman, Inge; Verhagen, Lennart; Overeem, Sebastiaan; van Elswijk, Gijs; Bloem, Bas; Hagoort, Peter; Toni, Ivan

    2014-02-01

    Despite the ambiguity inherent in human communication, people are remarkably efficient in establishing mutual understanding. Studying how people communicate in novel settings provides a window into the mechanisms supporting the human competence to rapidly generate and understand novel shared symbols, a fundamental property of human communication. Previous work indicates that the right posterior superior temporal sulcus (pSTS) is involved when people understand the intended meaning of novel communicative actions. Here, we set out to test whether normal functioning of this cerebral structure is required for understanding novel communicative actions using inhibitory low-frequency repetitive transcranial magnetic stimulation (rTMS). A factorial experimental design contrasted two tightly matched stimulation sites (right pSTS vs left MT+, i.e., a contiguous homotopic task-relevant region) and tasks (a communicative task vs a visual tracking task that used the same sequences of stimuli). Overall task performance was not affected by rTMS, whereas changes in task performance over time were disrupted according to TMS site and task combinations. Namely, rTMS over pSTS led to a diminished ability to improve action understanding on the basis of recent communicative history, while rTMS over MT+ perturbed improvement in visual tracking over trials. These findings qualify the contributions of the right pSTS to human communicative abilities, showing that this region might be necessary for incorporating previous knowledge, accumulated during interactions with a communicative partner, to constrain the inferential process that leads to action understanding. PMID:24268321

  10. Social Action As An Objective of Social Studies Instruction.

    ERIC Educational Resources Information Center

    Curtis, Charles K.

    This paper presents a rationale for making social action a major goal of elementary and secondary school social studies education. In addition, it describes social action models, suggests social action approaches appropriate for students at various grade levels, and reviews literature on social action by public school students. Social action is…

  11. Period and light-curve study of the contact eclipsing binary V523 Cas

    NASA Astrophysics Data System (ADS)

    Mohammadi, Mahya; Abedi, Abbas; Riazi, Nematollah

    2016-04-01

    CCD photometry of the eclipsing W Uma binary system V523 Cas in U, B, V and RC filters was carried out during eight nights in 2012. The physical and geometrical parameters of this system are obtained. A possible pulsation period of one of the components is obtained by analyzing the residuals of the ephemeris light curve. Our observations contain 16 times of minimum light. We combined these with all available published times of minimum. By fitting a quadratic curve to the O-C values, a new ephemeris of the system is calculated. By attributing the period change to mass transfer, we find a mass transfer rate of 4 ×10-12 M⊙/yr. Also, Period (80.58 yr) and the minimum mass (0.3 M⊙) of a possible third body is estimated. In addition, the possible existence of a fourth body with a mass of order 0.15 M⊙ is discussed. These third and fourth bodies could be low-mass main-sequence stars (red dwarfs).

  12. X-ray Studies of Radioactivity in the Tycho and Cas A SNR

    NASA Astrophysics Data System (ADS)

    Ryle, W. T.; Leising, M. D.

    2002-12-01

    A direct method for tracing isotopic abundances created through supernovae involves the measure of X-ray lines from radioactive isotopes formed in a supernova explosion. These X-rays originate from electron vacancies created by the electron-capture decay of proton rich isotopes produced in the supernova explosions. We use Chandra X-ray Observatory data to search for the lines from the decay of 59Ni and 44Ti isotopes in the Tycho supernova remnant (SNR) and the Cas A SNR, respectively. Using CIAO data analysis software, we determine only upper limits for the fluxes of the 6.90 keV 59Ni line and 4.09 keV 44Ti line, as we find no significant flux in either line. We present the limits obtained, which are consistent with theoretical estimates, and argue that these justify further observations of both SNR's with current X-ray observatories. This work was supported by the National Science Foundation through grant AST-0097616 for the SARA Research Experiences for Undergraduates program, and by NASA NAG 5-10764.

  13. The Co-Emergence of Machine Techniques, Paper-and-Pencil Techniques, and Theoretical Reflection: A Study of CAS Use in Secondary School Algebra

    ERIC Educational Resources Information Center

    Kieran, Carolyn; Drijvers, Paul

    2006-01-01

    This paper addresses the dialectical relation between theoretical thinking and technique, as they co-emerge in a combined computer algebra (CAS) and paper-and-pencil environment. The theoretical framework in this ongoing study consists of the instrumental approach to tool use and an adaptation of Chevallard's anthropological theory. The main aim…

  14. Short-term temporal studies of the X ray emission from Cas A, Tycho and Sco X-1

    NASA Technical Reports Server (NTRS)

    Holt, S. S.; Boldt, E. A.; Brisken, A. F.; Serlemitsos, P. J.

    1972-01-01

    No evidence for stable 2-10 keV periodic emission from Cas A or Tycho in the period range 1 msec to 10 sec is found. Upper limits to the pulsed fraction are presented as a function of the assumed light curve, with absolute 99% confidence upper limits of 0.089 and 0.195 for Cas A and Tycho, respectively. Previously reported transient 1-10 Hz oscillations from Sco X-1 are not observed.

  15. Interacting Parallel Constructions of Knowledge in a CAS Context

    ERIC Educational Resources Information Center

    Kidron, Ivy; Dreyfus, Tommy

    2010-01-01

    We consider the influence of a CAS context on a learner's process of constructing a justification for the bifurcations in a logistic dynamical process. We describe how instrumentation led to cognitive constructions and how the roles of the learner and the CAS intertwine, especially close to the branching and combining of constructing actions. The…

  16. The Impact on Student Achievement of When CAS Technology Is Introduced

    ERIC Educational Resources Information Center

    Driver, David

    2012-01-01

    When a Computer Algebra System (CAS) is used as a pedagogical and functional tool in class and as a functional tool in exams, its effect on student achievement can be quite profound. The timing of when students are first introduced to a CAS has an impact on gains in student achievement. In this action research project, the CAS calculator was…

  17. 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…

  18. 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. PMID:26929403

  19. [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…

  20. Functional annotation of native enhancers with a Cas9-histone demethylase fusion.

    PubMed

    Kearns, Nicola A; Pham, Hannah; Tabak, Barbara; Genga, Ryan M; Silverstein, Noah J; Garber, Manuel; Maehr, René

    2015-05-01

    Understanding of mammalian enhancers is limited by the lack of a technology to rapidly and thoroughly test the cell type-specific function. Here, we use a nuclease-deficient Cas9 (dCas9)-histone demethylase fusion to functionally characterize previously described and new enhancer elements for their roles in the embryonic stem cell state. Further, we distinguish the mechanism of action of dCas9-LSD1 at enhancers from previous dCas9-effectors. PMID:25775043

  1. Dance Education Action Research: A Twin Study

    ERIC Educational Resources Information Center

    Giguere, Miriam

    2015-01-01

    In this article, the author compares the practices, philosophy, and history of action research, also known as participatory action research, to the purposes and practices of dance education. The comparison yields connections in four categories, enhancing self-reflective teaching and curriculum design, taking responsibility for teaching outcomes,…

  2. A study of actions in operative notes.

    PubMed

    Wang, Yan; Pakhomov, Serguei; Burkart, Nora E; Ryan, James O; Melton, Genevieve B

    2012-01-01

    Operative notes contain rich information about techniques, instruments, and materials used in procedures. To assist development of effective information extraction (IE) techniques for operative notes, we investigated the sublanguage used to describe actions within the operative report 'procedure description' section. Deep parsing results of 362,310 operative notes with an expanded Stanford parser using the SPECIALIST Lexicon resulted in 200 verbs (92% coverage) including 147 action verbs. Nominal action predicates for each action verb were gathered from WordNet, SPECIALIST Lexicon, New Oxford American Dictionary and Stedman's Medical Dictionary. Coverage gaps were seen in existing lexical, domain, and semantic resources (Unified Medical Language System (UMLS) Metathesaurus, SPECIALIST Lexicon, WordNet and FrameNet). Our findings demonstrate the need to construct surgical domain-specific semantic resources for IE from operative notes. PMID:23304423

  3. 75 FR 66822 - Study on Extraterritorial Private Rights of Action

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-29

    ... COMMISSION Study on Extraterritorial Private Rights of Action AGENCY: Securities and Exchange Commission. ACTION: Request for Comments. SUMMARY: Section 929Y of the Dodd-Frank Wall Street Reform and Consumer... rights of action under the antifraud provisions of the Securities Exchange Act of 1934 (the...

  4. A database of whole-body action videos for the study of action, emotion, and untrustworthiness.

    PubMed

    Keefe, Bruce D; Villing, Matthias; Racey, Chris; Strong, Samantha L; Wincenciak, Joanna; Barraclough, Nick E

    2014-12-01

    We present a database of high-definition (HD) videos for the study of traits inferred from whole-body actions. Twenty-nine actors (19 female) were filmed performing different actions-walking, picking up a box, putting down a box, jumping, sitting down, and standing and acting-while conveying different traits, including four emotions (anger, fear, happiness, sadness), untrustworthiness, and neutral, where no specific trait was conveyed. For the actions conveying the four emotions and untrustworthiness, the actions were filmed multiple times, with the actor conveying the traits with different levels of intensity. In total, we made 2,783 action videos (in both two-dimensional and three-dimensional format), each lasting 7 s with a frame rate of 50 fps. All videos were filmed in a green-screen studio in order to isolate the action information from all contextual detail and to provide a flexible stimulus set for future use. In order to validate the traits conveyed by each action, we asked participants to rate each of the actions corresponding to the trait that the actor portrayed in the two-dimensional videos. To provide a useful database of stimuli of multiple actions conveying multiple traits, each video name contains information on the gender of the actor, the action executed, the trait conveyed, and the rating of its perceived intensity. All videos can be downloaded free at the following address: http://www-users.york.ac.uk/~neb506/databases.html. We discuss potential uses for the database in the analysis of the perception of whole-body actions. PMID:24584971

  5. Substrate generation for endonucleases of CRISPR/cas systems.

    PubMed

    Zoephel, Judith; Dwarakanath, Srivatsa; Richter, Hagen; Plagens, André; Randau, Lennart

    2012-01-01

    The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) (1-3). Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems (4). The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster (5). The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs (6-8). These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence (9). A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity(10). Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA (11,12) . These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases (4). Here, we present methods to generate crRNAs and precursor-cRNAs for

  6. Costs of CRISPR-Cas-mediated resistance in Streptococcus thermophilus

    PubMed Central

    Vale, Pedro F.; Lafforgue, Guillaume; Gatchitch, Francois; Gardan, Rozenn; Moineau, Sylvain; Gandon, Sylvain

    2015-01-01

    CRISPR-Cas is a form of adaptive sequence-specific immunity in microbes. This system offers unique opportunities for the study of coevolution between bacteria and their viral pathogens, bacteriophages. A full understanding of the coevolutionary dynamics of CRISPR-Cas requires knowing the magnitude of the cost of resisting infection. Here, using the gram-positive bacterium Streptococcus thermophilus and its associated virulent phage 2972, a well-established model system harbouring at least two type II functional CRISPR-Cas systems, we obtained different fitness measures based on growth assays in isolation or in pairwise competition. We measured the fitness cost associated with different components of this adaptive immune system: the cost of Cas protein expression, the constitutive cost of increasing immune memory through additional spacers, and the conditional costs of immunity during phage exposure. We found that Cas protein expression is particularly costly, as Cas-deficient mutants achieved higher competitive abilities than the wild-type strain with functional Cas proteins. Increasing immune memory by acquiring up to four phage-derived spacers was not associated with fitness costs. In addition, the activation of the CRISPR-Cas system during phage exposure induces significant but small fitness costs. Together these results suggest that the costs of the CRISPR-Cas system arise mainly due to the maintenance of the defence system. We discuss the implications of these results for the evolution of CRISPR-Cas-mediated immunity. PMID:26224708

  7. Efficient Mitochondrial Genome Editing by CRISPR/Cas9

    PubMed Central

    Jo, Areum; Ham, Sangwoo; Lee, Gum Hwa; Lee, Yun-Il; Kim, SangSeong; Lee, Yun-Song; Shin, Joo-Ho; Lee, Yunjong

    2015-01-01

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing. PMID:26448933

  8. Corrective Action Plan for Corrective Action Unit 271: Areas 25, 26, and 27 Septic Systems, Nevada Test Site, Nevada

    SciTech Connect

    R. B. Jackson

    2003-05-01

    The Areas 25, 26 and 27 Septic Systems are in the Federal Facility Agreement and Consent Order (FFACO) of 1996 as Corrective Action Unit (CAU) 271. This Corrective Action Plan (CAP) provides selected corrective action alternatives and proposes the closure methodology for CAU 271. CAU 271 is located on the Nevada Test Site (NTS) approximately 105 kilometers (65 miles) northwest of Las Vegas, Nevada, and consists of the following 15 Corrective Action Sites (CAS): CAS 25-04-1, Septic System; CAS 25-04-03, Septic System; CAS25-04-04, Septic System; CAS 25-04-08, Septic System; CAS 25-04-09, Septic System; CAS 25-04-10, Septic System; CAS 25-04-11, Septic System; CAS 26-03-01, Contaminated Water Reservoir; CAS 26-04-1, Septic System; CAS 26-04-02, Septic System; CAS 26-05-01, Radioactive Leachfield; CAS-26-05-03, Septic System; CAS 26-05-04, Septic System; CAS 26-05-05, Septic System; and CAS 27-05-02, Leachfield.

  9. Student Voice in High School: An Action Research Study

    ERIC Educational Resources Information Center

    Termini, Lorraine

    2013-01-01

    This action research study examined the effects of student voice in one high school and the self-reflection of the researcher-administrator involved in the effort. Using three cycles of action research, the researcher-administrator completed a pilot study, implemented a student voice project in one class, and developed a professional development…

  10. Middle School Responses to Cyberbullying: An Action Research Study

    ERIC Educational Resources Information Center

    Zidack, Astri Marie

    2013-01-01

    This action research study engaged a small public middle school in the northwest United States in a collaborative process to address cyberbullying issues that often lead to academic and behavior problems in schools (Hinduja, 2010; Olweus, 2010). The specific purpose of this action research study was to address the middle school's cyberbullying…

  11. Embodied Learning and Creative Writing: An Action Research Study

    ERIC Educational Resources Information Center

    Tobin, Jennifer Ann

    2012-01-01

    This action research study used narrative analysis to explore the role of the body in the writing process of creative writers. Specifically, the purpose of this action research study was threefold: it was first to examine how professional creative writers describe their writing process with particular attention to their perceptions of the role and…

  12. 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

  13. Advances in therapeutic CRISPR/Cas9 genome editing.

    PubMed

    Savić, Nataša; Schwank, Gerald

    2016-02-01

    Targeted nucleases are widely used as tools for genome editing. Two years ago the clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 nuclease was used for the first time, and since then has largely revolutionized the field. The tremendous success of the CRISPR/Cas9 genome editing tool is powered by the ease design principle of the guide RNA that targets Cas9 to the desired DNA locus, and by the high specificity and efficiency of CRISPR/Cas9-generated DNA breaks. Several studies recently used CRISPR/Cas9 to successfully modulate disease-causing alleles in vivo in animal models and ex vivo in somatic and induced pluripotent stem cells, raising hope for therapeutic genome editing in the clinics. In this review, we will summarize and discuss such preclinical CRISPR/Cas9 gene therapy reports. PMID:26470680

  14. Inducible in vivo genome editing with CRISPR/Cas9

    PubMed Central

    O'Rourke, Kevin P; Muley, Ashlesha; Kastenhuber, Edward R; Livshits, Geulah; Tschaharganeh, Darjus F; Socci, Nicholas D; Lowe, Scott W

    2015-01-01

    CRISPR/Cas9-based genome editing enables the rapid genetic manipulation of any genomic locus without the need for gene targeting by homologous recombination. Here we describe a conditional transgenic approach that allows temporal control of CRISPR/Cas9 activity for inducible genome editing in adult mice. We show that doxycycline-regulated Cas9 induction enables widespread gene disruption in multiple tissues and that limiting the duration of Cas9 expression or using a Cas9D10A (Cas9n) variant, can regulate the frequency and size of target gene modifications, respectively. Further, we show that the inducible CRISPR (iCRISPR) system can be used effectively to create biallelic mutation in multiple target loci and thus, provides a flexible and fast platform to study loss of function phenotypes in vivo. PMID:25690852

  15. Vertical spectrum of the C2H2+ system. An open shell (SC)2-CAS-SDCI study.

    PubMed

    Pitarch-Ruiz, José; Sánchez-Marín, José; Maynau, Daniel

    2003-04-15

    The open shell (SC)(2)-CAS-SDCI method along with a basis set of atomic natural orbitals (ANO) has been applied for calculating the main ionization potentials of acetylene, as well as the manifold of excited states of the different symmetries up to 32 eV. In this method, the single and double excitations of a CAS space are generated and the corresponding CI matrix is corrected by means of the (SC)(2) procedure that cancels the size-extensivity error and adds some high order contributions. The mean absolute error for the outer-valence X (2)Pi(u)(1pi(u) (-1)), A (2)Sigma(g) (+)(3sigma(g) (-1)), and B (2)Sigma(u) (+)(2sigma(u) (-1)) states, and the inner-valence C (2)Sigma(g) (+)(2sigma(g) (-1)) state is 0.1 eV. The excited states of C(2)H(2) (+) corresponding to the Sigma(g) (+), Sigma(u) (+), Pi(g), Pi(u), Delta(g), and Delta(u) symmetries are reported and their composition is discussed. The results are thoroughly compared to the best available multireference CI calculations. Recent multichannel CI results by Wells and Lucchese [J Chem Phys 1999, 110, 6365] have been used also as a guide for the discussion of the results. Discrepancies in the description of many multiconfigurational states by means of the (SC)(2)-CAS-SDCI wave function as compared to previous large MR-CI calculations are significant and are, consequently, remarked on. A large mixing of the 3sigma(g) (-1) and 2sigma(g) (-1) processes is found in the A (2)Sigma(g) (+) and C (2)Sigma(g) (+) states, provided that the basis set is augmented with Rydberg functions. PMID:12632475

  16. CRISPR/Cas9 Genome Editing as a Strategy to Study the Tumor Microenvironment in Transgenic Mice.

    PubMed

    Yamanaka, Yojiro

    2016-01-01

    Development of engineered site-specific endonucleases like zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 has been revolutionizing genetic approaches in biomedical research fields. These new tools have opened opportunities to carry out targeted genome editing in mouse zygotes without the need for manipulating embryonic stem cells, which have a higher technical burden and many constraints in strain availability. Specific genetic modifications can be directly generated in working genetic backgrounds. This new approach saves time and costs associated with generation and backcrossing of genetically modified animals and will facilitate their use in various cancer research fields. PMID:27581028

  17. Cas9 Functionally Opens Chromatin.

    PubMed

    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

  18. 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

  19. CRISPR-Cas9-guided Genome Engineering in C. elegans

    PubMed Central

    Kim, Hyun-Min; Colaiácovo, Monica P.

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is successfully being used for efficient and targeted genome editing in various organisms including the nematode C. elegans. Recent studies developed various CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: non-homologous end joining and homologous recombination. Here we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning and injection methods required for delivering Cas9, sgRNAs and repair template DNA into the C. elegans germline. PMID:27366893

  20. Studying Action Representation in Children via Motor Imagery

    ERIC Educational Resources Information Center

    Gabbard, Carl

    2009-01-01

    The use of motor imagery is a widely used experimental paradigm for the study of cognitive aspects of action planning and control in adults. Furthermore, there are indications that motor imagery provides a window into the process of action representation. These notions complement internal model theory suggesting that such representations allow…

  1. A Study of Educational Components of Community Action Programs.

    ERIC Educational Resources Information Center

    Computer Applications, Inc., Silver Spring, MD.

    Information was gathered concerning community Action Agencies (CAA) educational activities, programatic activities and organizational interactions both within the CAA structure and between the CAA and other organizations. The study was designed to focus on a comparison between CAA educational activities and specified Community Action Program (CAP)…

  2. Leadership development through action learning sets: an evaluation study.

    PubMed

    Walia, Surinder; Marks-Maran, Di

    2014-11-01

    This article examines the use of action learning sets in a leadership module delivered by a university in south east England. An evaluation research study was undertaking using survey method to evaluate student engagement with action learning sets, and their value, impact and sustainability. Data were collected through a questionnaire with a mix of Likert-style and open-ended questions and qualitative and quantitative data analysis was undertaken. Findings show that engagement in the action learning sets was very high. Action learning sets also had a positive impact on the development of leadership knowledge and skills and are highly valued by participants. It is likely that they would be sustainable as the majority would recommend action learning to colleagues and would consider taking another module that used action learning sets. When compared to existing literature on action learning, this study offers new insights as there is little empirical literature on student engagement with action learning sets and even less on value and sustainability. PMID:25164108

  3. Democracy and Diversity: Social Studies in Action

    ERIC Educational Resources Information Center

    Garcia, Jesus

    2005-01-01

    The author of this article, president of NCSS (2004-2005), is a professor of social studies education at the University of Nevada, Las Vegas. This address was delivered at the 84th NCSS Annual Conference in Baltimore, Maryland, November 2004. The author's vision for social studies and NCSS in the twenty-first century comes from growing up in a…

  4. An ERP study of motor compatibility effects in action language.

    PubMed

    Santana, Eduardo J; de Vega, Manuel

    2013-08-14

    This ERP study explores the brain's response to the manipulation of motor compatibility in action-related language. In Experiment 1 participants read sentences in which a protagonist performed two different manual actions either simultaneously or consecutively (e.g. While/after cleaning the wound he unrolled the bandage…). The ERPs were measured in the second-clause verb (e.g. unrolled) and noun (e.g. bandage). Notably, only the noun showed compatibility effects, namely a larger N400 in the simultaneous (incompatible) version than in the consecutive (compatible) version, suggesting that readers need to integrate the meaning of the whole sentence to evaluate the feasibility of the actions. In Experiment 2, motor compatibility was manipulated in a different way: all the sentences described the protagonist as performing two simultaneous actions that were both manual (While cleaning the wound he unrolled the bandage), or one action that was perceptual and the other manual (While looking at the wound he unrolled the bandage). The N400 effects for the former incompatible condition were replicated, again in the second-clause noun. The results demonstrated that readers of action language employ their pragmatic world knowledge to test the feasibility of motor actions, taking into account the embodied constraints of such actions. PMID:23796780

  5. Crystallization and preliminary X-ray crystallographic studies of the ArsI C-As lyase from Thermomonospora curvata.

    PubMed

    Nadar, S Venkadesh; Yoshinaga, Masafumi; Kandavelu, Palani; Sankaran, Banumathi; Rosen, Barry P

    2014-06-01

    Arsenic is a ubiquitous and carcinogenic environmental element that enters the biosphere primarily from geochemical sources, but also through anthropogenic activities. Microorganisms play an important role in the arsenic biogeochemical cycle by biotransformation of inorganic arsenic into organic arsenicals and vice versa. ArsI is a microbial nonheme ferrous-dependent dioxygenase that transforms toxic methylarsonous acid to the less toxic inorganic arsenite by C-As bond cleavage. An ArsI ortholog from the thermophilic bacterium Thermomonospora curvata was expressed, purified and crystallized. The crystals diffracted to 1.46 Å resolution and belonged to space group P4₃2₁2 or its enantiomer P4₁2₁2, with unit-cell parameters a=b=42.2, c=118.5 Å. PMID:24915088

  6. Functional annotation of native enhancers with a Cas9 -histone demethylase fusion

    PubMed Central

    Tabak, Barbara; Genga, Ryan M; Silverstein, Noah J; Garber, Manuel; Maehr, René

    2015-01-01

    Understanding of mammalian enhancer function is limited by the lack of a technology to rapidly and thoroughly test their cell type-specific function. Here, we use a nuclease-deficient (d)Cas9 histone demethylase fusion to functionally characterize previously described and novel enhancer elements for their roles in the embryonic stem cell state. Further, we distinguish the mechanism of action of dCas9-LSD1 at enhancers from previous dCas9-effectors. PMID:25775043

  7. Professional Vision in Action: An Exploratory Study

    ERIC Educational Resources Information Center

    Sherin, Miriam Gamoran; Russ, Rosemary S.; Sherin, Bruce L.; Colestock, Adam

    2008-01-01

    The study of teachers' professional vision poses some unique challenges. The application of professional vision happens in a manner that is fleeting, and that is distributed through the moments of instruction. Because of the ongoing nature of instruction, it is not realistic to expect that one could "pause" instruction momentarily, ask a teacher…

  8. Engineering a Poem: An Action Research Study

    ERIC Educational Resources Information Center

    Koch, Janice; Feingold, Brooke

    2006-01-01

    This study explores the use of design technology to teach a unit on poetry in a fifth grade class. The main goals of the poetry unit were to develop students' abilities to use their own creative voices to express themselves and to write descriptive poetry that creates detailed images for the reader. This unit was implemented in a fifth grade class…

  9. Studying Distance Students: Methods, Findings, Actions

    ERIC Educational Resources Information Center

    Wahl, Diane; Avery, Beth; Henry, Lisa

    2013-01-01

    University of North Texas (UNT) Libraries began studying the library needs of distance learners in 2009 using a variety of approaches to explore and confirm these needs as well as obtain input into how to meet them. Approaches used to date include analysis of both quantitative and qualitative responses by online students to the LibQUAL+[R] surveys…

  10. New Case Studies of Citizen Action

    ERIC Educational Resources Information Center

    Tanner, Thomas

    1977-01-01

    Describes a six-unit case study curriculum package designed for secondary and college-level courses relating to environmental education. The units deal with nuclear power, stream channelization, a river dam project, overgrazing of public lands, agribusiness versus the family farm, and swamp preservation. (Author/DB)

  11. From Calculus to Dynamical Systems through DGS and CAS

    ERIC Educational Resources Information Center

    García, Jeanett López; Zamudio, Jorge Javier Jiménez

    2015-01-01

    Several factors have motivated the use of CAS or DGS in the teaching-learning process, such as: the development of new technologies, the availability of computers, and the widespread use of the Internet, among others. Even more, the trend to include CAS and DGS in the curricula of some undergraduate studies has resulted in the instruction of the…

  12. [Epilobium parviflorum--in vitro study of biological action].

    PubMed

    Hevesi Tóth, Barbara; Kéry, Agnes

    2009-01-01

    Epilobium parviflorum Schreb. (willow-herb) is used for the treatment of benign prostatic hyperplasia (BPH), but its biological action is not entirely identified. This paper aims to report data on willow-herbs probable biological effect. In vitro studies have been made to investigate different aspects of its antioxidant capacity, anti-inflammatory (COX-inhibitory) action, steroid-receptor -agonistic and -antagonostic effect as well as aromatase-inhibitory effect. Based on our results, willow-herb possess remarkable antioxidant and COX-inhibitory action. PMID:19526676

  13. Cas9 Variants Expand the Target Repertoire in Caenorhabditis elegans.

    PubMed

    Bell, Ryan T; Fu, Becky X H; Fire, Andrew Z

    2016-02-01

    The proliferation of CRISPR/Cas9-based methods in Caenorhabditis elegans has enabled efficient genome editing and precise genomic tethering of Cas9 fusion proteins. Experimental designs using CRISPR/Cas9 are currently limited by the need for a protospacer adjacent motif (PAM) in the target with the sequence NGG. Here we report the characterization of two modified Cas9 proteins in C. elegans that recognize NGA and NGCG PAMs. We found that each variant could stimulate homologous recombination with a donor template at multiple loci and that PAM specificity was comparable to that of wild-type Cas9. To directly compare effectiveness, we used CRISPR/Cas9 genome editing to generate a set of assay strains with a common single-guide RNA (sgRNA) target sequence, but that differ in the juxtaposed PAM (NGG, NGA, or NGCG). In this controlled setting, we determined that the NGA PAM Cas9 variant can be as effective as wild-type Cas9. We similarly edited a genomic target to study the influence of the base following the NGA PAM. Using four strains with four NGAN PAMs differing only at the fourth position and adjacent to the same sgRNA target, we observed that efficient homologous replacement was attainable with any base in the fourth position, with an NGAG PAM being the most effective. In addition to demonstrating the utility of two Cas9 mutants in C. elegans and providing reagents that permit CRISPR/Cas9 experiments with fewer restrictions on potential targets, we established a means to benchmark the efficiency of different Cas9::PAM combinations that avoids variations owing to differences in the sgRNA sequence. PMID:26680661

  14. On the Integration of Computer Algebra Systems (CAS) by Canadian Mathematicians: Results of a National Survey

    ERIC Educational Resources Information Center

    Buteau, Chantal; Jarvis, Daniel H.; Lavicza, Zsolt

    2014-01-01

    In this article, we outline the findings of a Canadian survey study (N = 302) that focused on the extent of computer algebra systems (CAS)-based technology use in postsecondary mathematics instruction. Results suggest that a considerable number of Canadian mathematicians use CAS in research and teaching. CAS use in research was found to be the…

  15. After 16 Years of Publishing Standards, Do CAS Standards Make a Difference?

    ERIC Educational Resources Information Center

    Arminio, Jan; Gochenauer, Patty

    2004-01-01

    Using members of professional associations who are a part of the Council for the Advancement of Standards in Higher Education (CAS) consortia as a sample, this study investigated who uses CAS Standards, how and why they are used, and whether CAS Standards are associated with enhanced student learning. Using a quantitative analysis, this study…

  16. A Study of SN Ejecta in the Core-Collapse Supernova Remnant G292.0+1.8: Cas A's Older Cousin

    NASA Astrophysics Data System (ADS)

    Fesen, Robert

    2006-07-01

    Recent studies of the southern oxygen-rich supernova remnant {SNR} G292.0+1.8 have shown it to be the only Galactic SNR to exhibit all the features we expect in young remnants of core-collapse supernovae: an outer shell behind an expanding primary shock, high-velocity fragments of undiluted metal-rich ejecta, and a central pulsar surrounded by a pulsar-wind nebula. G292.0+1.8's optical emission consists of numerous knots and filaments of O- and S-rich ejecta spread throughout much of the remnant shell, many with radially oriented pencil-like geometries that may trace their origins to Rayleigh-Taylor instabilities during the SN event. The evolution and fine-scale structure of SN debris in young remnants is poorly understood and largely uncharted territory. For testing models for the distribution of metal-rich ejecta from core-collapse SNe, how the ejecta evolve and clump, and how SN shocks interact with the local circumstellar medium, the 3000-yr-old G292.0+1.8 remnant rivals the 320-yr-old Cas A remnant in importance. We therefore propose the first HST images of G292.0+1.8 in order to characterize the fine-scale spatial distribution of the ejecta, their sub-arcsecond chemical make-up, and the detailed structure and scale lengths for metal-rich SN ejecta clumps. The proposed HST images of G292.0+1.8 will be used in conjunction with existing Spitzer Cycle 1 infrared data and an upcoming 0.5 Msec Chandra X-ray image. We expect to achieve the same kind of results for G292 that have already been obtained for Cas A. High-resolution HST images of this remnant, combined with Spitzer and Chandra data and contrasted with a similar data set on Cas A, will provide superb multiwavelength benchmarks for both very young and older core-collapse SNRs.

  17. Toxicology and Carcinogenesis Studies of Hexachloroethane (CAS No. 67-72-1) in F344/N Rats (Gavage Studies).

    PubMed

    1989-08-01

    Hexachloroethane is used in organic synthesis as a retarding agent in fermentation, as a camphor substitute in nitrocellulose, in pyrotechnics and smoke devices, in explosives, and as a solvent. In previous long-term gavage studies with B6C3F1 mice and Osbourne-Mendel rats (78 weeks of exposure followed by 12-34 weeks of observation), hexachloroethane caused increased incidences of hepatocellular carcinomas in mice. However, survival of low and high dose rats was reduced compared with that of vehicle controls, and the effects on rats were inconclusive. Therefore, additional toxicology and carcinogenesis studies were conducted in F344/N rats by administering hexachloroethane (approximately 99% pure) in corn oil by gavage to groups of males and females for 16 days, 13 weeks, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and in Chinese hamster ovary (CHO) cells. Urinalysis was performed in conjunction with the 13-week studies. Sixteen-Day Studies: In the 16-day studies (dose range, 187-3,000 mg/kg), all rats that received 1,500 or 3,000 mg/kg and 1/5 males and 2/5 females that received 750 mg/kg died before the end of the studies. Final mean body weights of rats that received 750 mg/kg were 25% lower than that of vehicle controls for males and 37% lower for females. Compound-related clinical signs seen at 750 mg/kg or more included dyspnea, ataxia, prostration, and excessive lacrimation. Other compound-related effects included hyaline droplet formation in the tubular epithelial cells in all dosed males and tubular cell regeneration and granular casts in the tubules at the corticomedullary junction in the kidney in males receiving 187 and 375 mg/kg. Thirteen-Week Studies: In the 13-week studies (dose range, 47-750 mg/kg), 5/10 male rats and 2/10 female rats that received 750 mg/kg died before the end of the studies. The final mean body weight of male rats that received 750 mg/kg was 19% lower than that of vehicle controls. Compound

  18. Conditional Control of CRISPR/Cas9 Function.

    PubMed

    Zhou, Wenyuan; Deiters, Alexander

    2016-04-25

    The recently discovered CRISPR/Cas9 endonuclease system, comprised of a guide RNA for the recognition of a DNA target and the Cas9 nuclease protein for binding and processing the target, has been extensively studied and has been widely applied in genome editing, synthetic biology, and transcriptional modulation in cells and animals. Toward more precise genomic modification and further expansion of the CRISPR/Cas9 system as a spatiotemporally controlled gene regulatory system, several approaches of conditional activation of Cas9 function using small molecules and light have recently been developed. These methods have led to improvements in the genome editing specificity of the CRISPR/Cas9 system and enabled its activation with temporal and spatial precision. PMID:26996256

  19. NTP Toxicology and Carcinogenesis Studies of Pentachlorophenol (CAS NO. 87-86-5) in F344/N Rats (Feed Studies).

    PubMed

    1999-04-01

    Pentachlorophenol has been used as an herbicide, algicide, defoliant, wood preservative, germicide, fungicide, and molluscicide. Pentachlorophenol was nominated by the National Cancer Institute for carcinogenicity testing based on its widespread use as a wood preservative, potential for entering the environment (pentachlorophenol residues have been found worldwide in soil, water, and air samples; in food products; and in human and animal tissues and body fluids), and likelihood of bioaccumulation in the environment (pentachlorophenol is persistent in soil, having a half-life of up to 5 years). Technical Report No. 349 contains the results of the 2-year studies of pentachlorophenol performed by the NTP with B6C3F1 mice. Male and female F344/N rats were exposed to pentachlorophenol (approximately 99% pure) in feed for 28 days or 2 years. Genetic toxicology studies were conducted in vitro in Salmonella typhimurium and cultured Chinese hamster ovary cells and in vivo in rat and mouse bone marrow cells. 28-DAY STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were given 0, 200, 400, 800, 1,600, or 3,200 ppm pentachlorophenol, equivalent to average daily doses of approximately 20, 40, 75, 150, or 270 mg pentachlorophenol/kg body weight to males and females in feed for 28 days. With the exception of one male and two females exposed to 3,200 ppm, all rats survived until the end of the study. The final mean body weights and body weight gains of male rats exposed to 1,600 or 3,200 ppm and female rats exposed to 400, 800, 1,600, or 3,200 ppm were significantly less than those of the controls; rats exposed to 3,200 ppm lost weight during the study. Feed consumption by 3,200 ppm males was less than that by the control group throughout the study. The absolute and relative liver weights of 400, 800, and 1,600 ppm males and all exposed groups of females were significantly greater than those of the controls. Compared to the control groups, the incidences of minimal to mild

  20. Toxicology and Carcinogenesis Studies of Quercetin (CAS No. 117-39-5) in F344 Rats (Feed Studies).

    PubMed

    1992-09-01

    Quercetin is a member of a group of naturally occurring compounds, the flavonoids, which have a common flavone nucleus composed of two benzene rings linked through a heterocyclicpyrone ring. Quercetin is found in various plants, food products, and dyes of natural origin. The estimated average daily intake of quercetin by an individual in the United States is 25 mg. The Food and Drug Administration nominated quercetin for toxicity and carcinogenicity studies in the rat because it is a chemical that is widely distributed in foods. Quercetin was administered to rats by dosed feed since human exposure is by dietary consumption. Information in the literature showed that quercetin administered in the diet to rats at levels up to approximately 4% caused a minor body weight effect, whereas higher dose levels produced greater than 10% reduction in body weight gains relative to controls. Based on this information, the NTP 2-year studies were conducted by administering 0, 1,000, 10,000, or 40,000 ppm quercetin (>95% pure) in feed to groups of 50 male and female rats for 104 weeks. Ten additional animals per dose group were evaluated at 6 and 15 months. Body Weight, Survival, and Clinical Findings in the 2-Year Studies: Body weights of exposed male and female rats given 1,000 and 10,000 ppm were within 5% of controls throughout the studies. Reduced body weight gain in male and female rats receiving 40,000 ppm was observed by week 15 and the final mean body weights were 87% of controls at week 104. Survival and feed consumption were similar among exposed and control groups throughout the studies. The average amounts of quercetin consumed per day by the 1,000, 10,000 and 40,000 ppm dose groups after week 52 were 40, 400, and 1,900 mg/kg of body weight. Nonneoplastic and Neoplastic Effects in the 2-Year Studies: In male rats, the principal toxic effects associated with the dietary administration of quercetin for 2 years were observed in the kidney. There were dose

  1. NTP Toxicology and Carcinogenesis Studies of Isoprene (CAS No. 78-79-5) in F344/N Rats (Inhalation Studies).

    PubMed

    1999-07-01

    Isoprene, the monomeric unit of natural rubber and naturally occurring terpenes and steroids, is primarily obtained as a by-product of naphtha cracking for ethylene production. It is emitted from plants and trees, has been detected in tobacco smoke and automobile exhaust, and was identified as a major endogenous hydrocarbon in human breath. Isoprene was selected for toxicologic evaluation because of its structural similarity to 1,3-butadiene, a potent, multi-organ, rodent carcinogen, and the potential for human exposure due to its large annual production volume. A previous 26-week inhalation study followed by a 26-week recovery period provided clear evidence of carcinogenic activity of isoprene in male B6C3F1 mice. A similar study in male F344/N rats was inconclusive. Male and female F344/N rats were exposed to isoprene (99% pure) by whole body inhalation for 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, mouse bone marrow and peripheral blood cells, and rat lung fibroblasts. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female F344/N rats were exposed to 220, 700, or 7,000 ppm isoprene by inhalation, 6 hours per day, 5 days per week, for 105 weeks. Survival and Body Weights: Survival rates and mean body weights of exposed male and female rats were similar to those of the chamber controls throughout the study. Urinary Vinyl Lactic Acid - Biomarker of Exposure: At 3, 6, 12, and 18 months, the concentrations of vinyl lactic acid normalized to creatinine in the urine increased with increasing exposure concentration in all exposed groups of male and female rats; however, these increases were not proportional to isoprene exposure concentrations, indicating nonlinear metabolism over this range of exposure concentrations. Pathology Findings: Exposure-related increases in the incidences of mam mary gland fibroadenoma were observed in male rats in all groups. The incidences of fibroadenoma in 7,000 ppm

  2. Distributed Leadership: Developing a New Practice. An Action Research Study

    ERIC Educational Resources Information Center

    Chamberland, Lorie

    2009-01-01

    This action research study examines the development of distributed leadership practice among a new team of teacher leaders and their principal at an elementary school. Using a qualitative approach including a series of interviews, observations and journal entries, this study analyzes the interactions and changing roles of teacher leaders and their…

  3. Historic light curve of V890 Cas

    NASA Astrophysics Data System (ADS)

    Nesci, R.

    2016-05-01

    The variability of V890 Cas is studied with 87 -band plates of the Asiago archive. The star shows variations of about 5 mag with an average magnitude =13 and a period of 486 days. An 5.0 color index is also derived near the maximum luminosity.

  4. 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.

  5. Lessons Learned on Management of CAS Development.

    ERIC Educational Resources Information Center

    Boyadjieff, Kiril

    1995-01-01

    Computer-assisted studies (CAS) attract foreign language professionals' attention due to the reliability of personal computers, the decreasing cost of available technology, and the new generation of students for whom electronic media are a familiar habitat. This article focuses on a project of the Defense Language Institute that produced over…

  6. 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. PMID:26535798

  7. Studies on the oxygenation of human blood by photocatalytic action.

    PubMed

    Subrahmanyam, Aryasomayajula; Arokiadoss, Thevasahayam; Ramesh, T Paul

    2007-11-01

    The present article gave the proof of concept for oxygenating human blood using the established principles of photocatalytic action of anatase TiO2 thin films in generating oxygen from water. The photocatalytic action involves the absorption of the UV optical energy (365 nm) to split water available in the blood into oxygen and hydrogen, and the generated oxygen is attached to the hemoglobin. In the present study, an enhanced catalytic action was achieved by preparing the nanosized anatase TiO2 thin films on tin-doped indium oxide (ITO) thin films, forming TiO2/ITO semiconducting junction. These TiO2 and ITO thin films and the semiconducting junctions were grown by the reactive DC Magnetron sputtering technique (using pure metallic targets) at room temperature (300 K) and subsequently annealed at 870 K for 60 min. The annealing process (i) influenced the formation of the anatase phase of TiO2; and (ii) diffused indium from ITO into TiO2, forming InTi(2)O(5). The work functions of ITO and InTi(2)O(5) were measured to be 4.72 and 4.76 eV, respectively. The higher efficacy of the photocatalytic action was attributed to the lower work function of ITO. The results clearly show that the photocatalytic action increases the oxygen content in the blood significantly. PMID:18001391

  8. Effective Use of Action-Oriented Studies in Pakistan.

    ERIC Educational Resources Information Center

    Miles, M.

    1991-01-01

    This article summarizes results from action-oriented studies carried out at the Mental Heath Centre in Peshawar, Pakistan, dealing with such topics as attitudes toward disability, casual integration, and trends in polio paralysis. The article also considers problems associated with the dissemination of special education and rehabilitation research…

  9. Study Challenges Assumptions about Affirmative-Action Bans

    ERIC Educational Resources Information Center

    Schmidt, Peter

    2008-01-01

    This article reports the results of a new study on the impact of bans on race-conscious admissions policies which seem to confirm what many critics of affirmative action have long suspected: It is Asian-Americans, rather than whites, who are most disadvantaged by elite universities' consideration of ethnicity and race. Left unanswered are the…

  10. Case Studies of Action Research in Various Adult Education Settings.

    ERIC Educational Resources Information Center

    Kuhne, Gary W.; Weirauch, Drucie; Fetterman, David J.; Mearns, Raiana M.; Kalinosky, Kathy; Cegles, Kathleen A.; Ritchey, Linda

    1997-01-01

    Six case studies illustrate action research in adult education: faculty development in a museum, participation in a church congregation, retention of literacy volunteers in a corrections center, learner participation in a homeless shelter, technology innovation in a university, and infection control in a hospital. (SK)

  11. A Longitudinal Study of the Impact of an Environmental Action

    ERIC Educational Resources Information Center

    Rioux, Liliane; Pasquier, Daniel

    2013-01-01

    In a previous study, we investigated the impact of an awareness-raising campaign on the behaviour of secondary school children in the Centre Region of France, regarding the recycling of used batteries. But, was it a question of pro-environmental behaviour or simply an environmental action? To answer this question, a three-year longitudinal study…

  12. Minesweeper and Hypothetical Thinking Action Research & Pilot Study

    ERIC Educational Resources Information Center

    Walker, Jacob J.

    2010-01-01

    This Action Research project and Pilot Study was designed and implemented to improve students' hypothetical thinking abilities by exploring the possibility that learning and playing the computer game Minesweeper may inherently help improve hypothetical thinking. One objective was to use educational tools to make it easier for students to learn the…

  13. A diary study of action slips in healthy individuals.

    PubMed

    Jónsdóttir, María K; Adólfsdóttir, Steinunn; Cortez, Rúna Dögg; Gunnarsdóttir, María; Gústafsdóttir, Agústa Hlín

    2007-12-01

    Memory complaints following minor head injury or whiplash are common and often bear similarity to absent mindedness or action slips (Reason, 1979). We replicated Reason's study by asking 189 healthy volunteers to keep diaries of their action slips for a week. The mean number of slips was 6.4 (SD = 4.9). Perceived stress did not correlate with number of slips but there was a weak positive correlation between action slips and scores on a memory failures questionnaire. Memory diaries may be clinically useful when assessing individuals who worry about cognitive sequelae of minor injuries. Diaries clarify the nature of the complaints and may have therapeutic value by demonstrating that the memory slips are less frequent than estimated by the patients. PMID:17853144

  14. Students of Action? A Comparative Investigation of Secondary Science and Social Studies Students' Action Repertoires in a Land Use Context

    ERIC Educational Resources Information Center

    Kumler, Lori M.

    2011-01-01

    Environmental education (EE) and social studies education share an interest in behavioral outcomes. This study compares behavioral outcomes--including both self-reported knowledge of actions and reported actions taken--in the context of a land use curriculum enacted in secondary science versus social studies classes with 500 students and nine…

  15. Genome Editing with CRISPR-Cas9: Can It Get Any Better?

    PubMed

    Haeussler, Maximilian; Concordet, Jean-Paul

    2016-05-20

    The CRISPR-Cas revolution is taking place in virtually all fields of life sciences. Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient, relying only on the design of a synthetic single guide RNA (sgRNA) and its co-expression with Cas9. Here, we review the progress in the design of sgRNA from the original dual RNA guide for S. pyogenes and Staphylococcus aureus Cas9 (SpCas9 and SaCas9). New assays for genome-wide identification of off-targets have provided important insights into the issue of cleavage specificity in vivo. At the same time, the on-target activity of thousands of guides has been determined. These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences. It appears that for most basic research applications, cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions. Moreover, recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing. Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA, several variants of SpCas9 have recently been engineered, either with novel protospacer adjacent motifs (PAMs) or with drastically reduced off-targets. Novel Cas9 and Cas9-like proteins called Cpf1 have also been characterized from other bacteria and will benefit from the insights obtained from SpCas9. Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage. PMID:27210042

  16. CRISPR/Cas9 in Genome Editing and Beyond.

    PubMed

    Wang, Haifeng; La Russa, Marie; Qi, Lei S

    2016-06-01

    The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers. PMID:27145843

  17. CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani

    PubMed Central

    Zhang, Wen-Wei

    2015-01-01

    ABSTRACT The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani. PMID:26199327

  18. Determination of the Physical Dimensions of μ Cas

    NASA Astrophysics Data System (ADS)

    Bach, K.; Kang, W.

    2015-07-01

    We investigate the physical properties of the nearby astrometric binary μ Cas based on a spectroscopic analysis and evolutionary calculations. The chemical composition of the μ Cas system has been determined based on high resolution spectroscopy from BOES. Combining our spectroscopic analysis with observations from Hipparcos and CHARA, we determine the evolutionary status of μ Cas. With well-constrained stellar parameters, the internal structure and frequency modes have also been calculated. The ultimate goal of this study is to describe the physical processes inside stars through a comprehensive modeling.

  19. 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.

  20. Action Researchers' Perspectives about the Distinguishing Characteristics of Action Research: A Delphi and Learning Circles Mixed-Methods Study

    ERIC Educational Resources Information Center

    Rowell, Lonnie L.; Polush, Elena Yu; Riel, Margaret; Bruewer, Aaron

    2015-01-01

    The purpose of this study was to identify distinguishing characteristics of action research within the Action Research Special Interest Group of the American Educational Research Association. The authors sought to delineate the foundational framework endorsed by this community. The study was conducted during January-April 2012 and employed an…

  1. Closure Report for Corrective Action Unit 107: Low Impact Soil Sites, Nevada Test Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2009-06-01

    Corrective Action Unit (CAU) 107 is identified in the Federal Facility Agreement and Consent Order (FFACO) as 'Low Impact Soil Sites' and consists of the following 15 Corrective Action Sites (CASs), located in Areas 1, 2, 3, 4, 5, 9, 10, and 18 of the Nevada Test Site: CAS 01-23-02, Atmospheric Test Site - High Alt; CAS 02-23-02, Contaminated Areas (2); CAS 02-23-03, Contaminated Berm; CAS 02-23-10, Gourd-Amber Contamination Area; CAS 02-23-11, Sappho Contamination Area; CAS 02-23-12, Scuttle Contamination Area; CAS 03-23-24, Seaweed B Contamination Area; CAS 03-23-27, Adze Contamination Area; CAS 03-23-28, Manzanas Contamination Area; CAS 03-23-29, Truchas-Chamisal Contamination Area; CAS 04-23-02, Atmospheric Test Site T4-a; CAS 05-23-06, Atmospheric Test Site; CAS 09-23-06, Mound of Contaminated Soil; CAS 10-23-04, Atmospheric Test Site M-10; and CAS 18-23-02, U-18d Crater (Sulky). Closure activities were conducted from February through April 2009 according to the FFACO (1996; as amended February 2008) and Revision 1 of the Streamlined Approach for Environmental Restoration Plan for CAU 107 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office, 2009). The corrective action alternatives included No Further Action and Closure in Place with Administrative Controls. Closure activities are summarized.

  2. Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9.

    PubMed

    Hirano, Seiichi; Nishimasu, Hiroshi; Ishitani, Ryuichiro; Nureki, Osamu

    2016-03-17

    The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets bearing a PAM (protospacer adjacent motif) and complementarity to the guide RNA. A recent study showed that, whereas wild-type Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5'-NGG-3' PAM, the engineered VQR, EQR, and VRER SpCas9 variants recognize the 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' PAMs, respectively, thus expanding the targetable sequences in Cas9-mediated genome editing applications. Here, we present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. Our findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9. PMID:26990991

  3. 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. PMID:26416740

  4. Cell transfection as a tool to study growth hormone action

    SciTech Connect

    Norstedt, G.; Enberg, B.; Francis, S.

    1994-12-31

    The isolation of growth hormone receptor (GHR) cDNA clones has made possible the transfection of GHRs into cultured cells. Our aim in this minireview is to show how the application of such approaches have benefited GHR research. GH stimulation of cells expressing GHR cDNAs can cause an alteration of cellular function that mimic those of the endogenous GHR. GHR cDNA transfected cells also offer a system where the mechanism of GH action can be studied. Such a system has been used to demonstrate that the GHR itself becomes tyrosine phosphorylated and that further phosphorylation of downstream proteins is important in GH action. The GH signals are transmitted to the nucleus and GH regulated genes have now begun to be characterized. The ability to use cell transfection for mechanistic studies of GH action will be instrumental to define domains within the receptor that are of functional importance and to determined pathways whereby GH signals are conveyed within the cell. 33 refs., 2 tabs.

  5. Rescue of the skeletal phenotype in CasR-deficient mice by transfer onto the Gcm2 null background

    PubMed Central

    Tu, Qisheng; Pi, Min; Karsenty, Gerard; Simpson, Leigh; Liu, Shiguang; Quarles, L. Darryl

    2003-01-01

    To understand the role of the calcium-sensing receptor (CasR) in the skeleton, we used a genetic approach to ablate parathyroid glands and remove the confounding effects of elevated parathyroid hormone (PTH) in CasR-deficient mice. CasR deficiency was transferred onto the glial cells missing 2–deficient (Gcm2-deficient) background by intercrossing CasR- and Gcm2-deficient mice. Superimposed Gcm2 deficiency rescued the perinatal lethality in CasR-deficient mice in association with ablation of the parathyroid glands and correction of the severe hyperparathyroidism. In addition, the double homozygous CasR- and Gcm2-deficient mice demonstrated healing of the abnormal mineralization of cartilage and bone associated with CasR deficiency, indicating that rickets and osteomalacia in CasR-deficient mice are not due to an independent function of CasR in bone and cartilage but to the effect of severe hyperparathyroidism in the neonate. Analysis of the skeleton of 6-week-old homozygous CasR- and Gcm2-deficient mice also failed to identify any essential, nonredundant role for CasR in regulating chondrogenesis or osteogenesis, but further studies are needed to establish the function of CasR in the skeleton. In contrast, concomitant Gcm2 and CasR deficiency failed to rescue the hypocalciuria in CasR-deficient mice, consistent with direct regulation of urinary calcium excretion by CasR in the kidney. Double Gcm2- and CasR-deficient mice provide an important model for evaluating the extraparathyroid functions of CasR. PMID:12671052

  6. Corrective Action Investigation Plan for Corrective Action Unit 106: Areas 5, 11 Frenchman Flat Atmospheric Sites, Nevada National Security Site, Nevada

    SciTech Connect

    Patrick Matthews

    2011-07-01

    Corrective Action Unit 106 comprises the four corrective action sites (CASs) listed below: • 05-20-02, Evaporation Pond • 05-23-05, Atmospheric Test Site - Able • 05-45-04, 306 GZ Rad Contaminated Area • 05-45-05, 307 GZ Rad Contaminated Area These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives (CAAs). Additional information will be obtained by conducting a corrective action investigation before evaluating CAAs and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viable CAAs that will be presented in the Corrective Action Decision Document. The sites will be investigated based on the data quality objectives (DQOs) developed on January 19, 2010, by representatives of the Nevada Division of Environmental Protection and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective actions for CAU 106. The presence and nature of contamination at CAU 106 will be evaluated based on information collected from a field investigation. The CAU includes land areas impacted by the release of radionuclides from groundwater pumping during the Radionuclide Migration study program (CAS 05-20-02), a weapons-related airdrop test (CAS 05-23-05), and unknown support activities at two sites (CAS 05-45-04 and CAS 05-45-05). The presence and nature of contamination from surface-deposited radiological contamination from CAS 05-23-05, Atmospheric Test Site - Able, and other types of releases (such as migration and excavation as well as any potential releases discovered during the investigation) from the remaining three CASs will be evaluated using soil samples collected from the locations

  7. Priorities for Action in a Rural Older Adults Study

    PubMed Central

    Averill, Jennifer B.

    2013-01-01

    This article reports the findings from a recent study of older adults in the rural southwestern United States and discusses practice and research implications. The aim of the study was to analyze health disparities and strengths in the contexts of rurality, aging, a depressed economy, and limited health resources. Identified themes needing action included sustained access to prescriptions, transportation solutions for older adults in isolated communities, inadequate access to care, poor infrastructure and coordination of services, scarce assisted living and in-home care for frail older adults, and barriers related to culture, language, and economics. PMID:22929381

  8. The Structural Biology of CRISPR-Cas Systems

    PubMed Central

    Jiang, Fuguo; Doudna, Jennifer A.

    2015-01-01

    Prokaryotic CRISPR-Cas genomic loci encode RNA-mediated adaptive immune systems that bear some functional similarities with eukaryotic RNA interference. Acquired and heritable immunity against bacteriophage and plasmids begins with integration of ~30 base pair foreign DNA sequences into the host genome. CRISPR-derived transcripts assemble with CRISPR-associated (Cas) proteins to target complementary nucleic acids for degradation. Here we review recent advances in the structural biology of these targeting complexes, with a focus on structural studies of the multisubunit Type I CRISPR RNA-guided surveillance and the Cas9 DNA endonuclease found in Type II CRISPR-Cas systems. These complexes have distinct structures that are each capable of site-specific double-stranded DNA binding and local helix unwinding. PMID:25723899

  9. CRISPR-Cas9-Guided Genome Engineering in C. elegans.

    PubMed

    Kim, Hyun-Min; Colaiácovo, Monica P

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is successfully being used for efficient and targeted genome editing in various organisms, including the nematode C. elegans. Recent studies have developed various CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: non-homologous end joining and homologous recombination. Here we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning, as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the C. elegans germline. © 2016 by John Wiley & Sons, Inc. PMID:27366893

  10. Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses.

    PubMed

    Wang, Dan; Mou, Haiwei; Li, Shaoyong; Li, Yingxiang; Hough, Soren; Tran, Karen; Li, Jia; Yin, Hao; Anderson, Daniel G; Sontheimer, Erik J; Weng, Zhiping; Gao, Guangping; Xue, Wen

    2015-07-01

    CRISPR/Cas9 derived from the bacterial adaptive immunity pathway is a powerful tool for genome editing, but the safety profiles of in vivo delivered Cas9 (including host immune responses to the bacterial Cas9 protein) have not been comprehensively investigated in model organisms. Nonalcoholic steatohepatitis (NASH) is a prevalent human liver disease characterized by excessive fat accumulation in the liver. In this study, we used adenovirus (Ad) vector to deliver a Streptococcus pyogenes-derived Cas9 system (SpCas9) targeting Pten, a gene involved in NASH and a negative regulator of the PI3K-AKT pathway, in mouse liver. We found that the Ad vector mediated efficient Pten gene editing even in the presence of typical Ad vector-associated immunotoxicity in the liver. Four months after vector infusion, mice receiving the Pten gene-editing Ad vector showed massive hepatomegaly and features of NASH, consistent with the phenotypes following Cre-loxP-induced Pten deficiency in mouse liver. We also detected induction of humoral immunity against SpCas9 and the potential presence of an SpCas9-specific cellular immune response. Our findings provide a strategy to model human liver diseases in mice and highlight the importance considering Cas9-specific immune responses in future translational studies involving in vivo delivery of CRISPR/Cas9. PMID:26086867

  11. Goal anticipation during action observation is influenced by synonymous action capabilities, a puzzling developmental study.

    PubMed

    Gredebäck, Gustaf; Kochukhova, Olga

    2010-04-01

    Eighteen- and 25-month-old human toddlers' ability to manually solve a puzzle and their ability to anticipate the goal during observation of similar actions were investigated. Results demonstrate that goal anticipation during action observation is dependent on manual ability, both on a group level (only 25-month-olds solved the manual task and anticipated the goal during observation) and individually within the older age group (r (xy) = 0.53). These findings suggests a connection between manual ability and the ability to anticipate the goal of others' actions in toddlers, in accordance with the direct matching hypothesis. PMID:20041233

  12. An Approach to the Study of Systems of Equations with Geogebra: Learning Opportunities Provided by the Integration of CAS View: Story of a Workshop Experience with Teachers

    ERIC Educational Resources Information Center

    Alejandra, Almirón; Fernando, Bifano; Leonardo, Lupinacci

    2015-01-01

    Solving systems of equations at school, at least in Argentina, is usually a task that students are given as a series of techniques that "allow" them to find a solution. How to overcome educational obstacles that are generated from a fragmented approach of knowledge? What can DGS do, in particular the CAS environment? What epistemic and…

  13. 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. PMID:23439366

  14. A Study of Hypergiant Mass Loss in the Near-To-Mid Infrared: VY CMa, IRC +10420, mu Cep and rho Cas

    NASA Astrophysics Data System (ADS)

    Shenoy, Dinesh Prabhakar

    2016-01-01

    Stars of initial mass greater than 9 M_sun become red supergiants (RSGs), a short-lived stage during which they experience mass-loss that strongly influences their post-RSG evolution and end state. The highest luminosity RSGs, referred to here as hypergiants, experience episodic mass-loss whose mechanism remains poorly understood and motivates observations to help constrain it. This thesis studies mass loss from hypergiant stars with near-to-mid infrared imaging over a range of angular scales. The recent mass-loss history of the extreme red supergiant VY Canis Majoris and the warm hypergiant star IRC +10420 are studied at the sub-arcsecond scale with adaptive optics imaging and imaging polarimetry from 1 - 5 micron using LMIRCam on the Large Binocular Telescope (LBT) and MMT-Pol at the MMT Observatory. The nebular features of VY CMa are found to be highly polarized at 1.3 and 3.1 micron, with optically thick scattering required to reproduce the observed surface brightness. The flux of VY CMa's peculiar ``Southwest Clump'' is demonstrated to be due almost entirely to optically thick scattering, with little thermal emission, and with a lower limit mass of 5E-03 M_sun in this single feature. The imaging polarimetry of IRC +10420 at 2.2 micron resolves nebular emission with intrinsic polarization of 30%, with a high surface brightness indicating optically thick scattering largely in the plane of the sky. Using the polarimetry to constrain the scattered light emission, it is shown that the nebula's the emission is mostly thermal with a color temperature well above that for typical astrophysical dust. To probe further into hypergiants' history of mass-loss, mid-IR imaging with MMT/ MIRAC and SOFIA/FORCAST is used to study VY CMa, IRC +10420 and two additional hypergiants: the RSG mu Cep and the warm hypergiant rho Cas. Using DUSTY 1-D radiative transfer models, mu Cep's mass-loss rate is found to have declined by about a factor of 5 over a 13,000 history, ranging from 5E

  15. Corrective Action Plan for Corrective Action Unit 490: Station 44 Burn Area, Tonopah Test Range, Nevada

    SciTech Connect

    K. B. Campbell

    2002-04-01

    Corrective Action Unit (CAU) 490, Station 44 Burn Area is located on the Tonopah Test Range (TTR). CAU 490 is listed in the Federal Facility Agreement and Consent Order (FFACO, 1996) and includes for Corrective Action Sites (CASs): (1) Fire Training Area (CAS 03-56-001-03BA); (2) Station 44 Burn Area (CAS RG-56-001-RGBA); (3) Sandia Service Yard (CAS 03-58-001-03FN); and (4) Gun Propellant Burn Area (CAS 09-54-001-09L2).

  16. Closure Report for Corrective Action Unit 134: Aboveground Storage Tanks, Nevada Test Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2009-06-30

    Corrective Action Unit (CAU) 134 is identified in the Federal Facility Agreement and Consent Order (FFACO) as “Aboveground Storage Tanks” and consists of the following four Corrective Action Sites (CASs), located in Areas 3, 15, and 29 of the Nevada Test Site: · CAS 03-01-03, Aboveground Storage Tank · CAS 03-01-04, Tank · CAS 15-01-05, Aboveground Storage Tank · CAS 29-01-01, Hydrocarbon Stain

  17. A Self-Study of the Teaching of Action Research in a University Context

    ERIC Educational Resources Information Center

    Choi, Jung-ah

    2011-01-01

    Despite the potential benefits of action research, teaching action research in a university setting can present challenges. Analyzing my own experiences of teaching a university-based course on action research, this self-study investigates what my students (all classroom teachers) did and did not understand about action research and what hindered…

  18. Corrective Action Plan for Corrective Action Unit 562: Waste Systems, Nevada National Security Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2011-04-30

    This Corrective Action Plan has been prepared for Corrective Action Unit (CAU) 562, Waste Systems, in accordance with the Federal Facility Agreement and Consent Order (1996; as amended March 2010). CAU 562 consists of 13 Corrective Action Sites (CASs) located in Areas 2, 23, and 25 of the Nevada National Security Site. Site characterization activities were performed in 2009 and 2010, and the results are presented in Appendix A of the Corrective Action Decision Document for CAU 562. The scope of work required to implement the recommended closure alternatives is summarized. (1) CAS 02-26-11, Lead Shot, will be clean closed by removing shot. (2) CAS 02-44-02, Paint Spills and French Drain, will be clean closed by removing paint and contaminated soil. As a best management practice (BMP), asbestos tile will be removed. (3) CAS 02-59-01, Septic System, will be clean closed by removing septic tank contents. As a BMP, the septic tank will be removed. (4) CAS 02-60-01, Concrete Drain, contains no contaminants of concern (COCs) above action levels. No further action is required; however, as a BMP, the concrete drain will be removed. (5) CAS 02-60-02, French Drain, was clean closed. Corrective actions were completed during corrective action investigation activities. As a BMP, the drain grates and drain pipe will be removed. (6) CAS 02-60-03, Steam Cleaning Drain, will be clean closed by removing contaminated soil. As a BMP, the steam cleaning sump grate and outfall pipe will be removed. (7) CAS 02-60-04, French Drain, was clean closed. Corrective actions were completed during corrective action investigation activities. (8) CAS 02-60-05, French Drain, will be clean closed by removing contaminated soil. (9) CAS 02-60-06, French Drain, contains no COCs above action levels. No further action is required. (10) CAS 02-60-07, French Drain, requires no further action. The french drain identified in historical documentation was not located during corrective action investigation

  19. Closure Report for Corrective Action Unit 350: Miscellaneous Housekeeping Sites, Nevada Test Site, Nevada

    SciTech Connect

    R. B. Jackson

    2003-05-01

    This Closure Report documents the closure activities conducted for Corrective Action Unit (CAU) 350: Miscellaneous Housekeeping sites. CAU 350 is listed in Appendix III of the Federal Facility Agreement and Consent Order (FFACO) (FFACO, 1996) and consists of the following eight Corrective Action Sites (CASs) located in Areas 12 and 15 of the Nevada Test Site (NTS): CAS 12-26-01, Lead Shot; CAS 15-22-04, Drums(2); CAS 15-22-06, Drums(10); CAS 15-22-16, Drums(3); CAS 15-22-22, Hydrocarbon Impacted Soil; CAS 15-22-29, Drums(2); CAS 15-24-07, Batteries; and CAS 15-99-02, Gas Cylinder. Closure activities consisted of closing each CAS by removing debris and/or material, disposing of the generated waste, and verifying that each site was clean-closed by visual inspection and/or laboratory analysis of soil samples.

  20. Neuronal and immunological basis of action of antidepressants in chronic pain - clinical and experimental studies.

    PubMed

    Mika, Joanna; Zychowska, Magdalena; Makuch, Wioletta; Rojewska, Ewelina; Przewlocka, Barbara

    2013-01-01

    The current knowledge of the pharmacological actions of the tricyclic antidepressants (TCAs) has slowly evolved through their over 40-year history. Chronic pain represents one of the most important public health problems, and antidepressants are an essential part of the therapeutic strategy in addition to classical analgesics. This article reviews the available evidence on the efficacy and safety of antidepressants in chronic pain conditions; namely, headaches, low back pain, fibromyalgia, cancer pain and especially neuropathic pain. TCAs are traditionally the main type of depression medication used to treat chronic pain. Recently, new antidepressants were introduced into clinical use, with a significant reduction in side effects and equivalent efficacy on mood disorders. These new drugs that are effective for chronic pain belong to the tetracyclic antidepressants (TeCAs) group (amoxapine, maprotiline), the serotonin and noradrenaline reuptake inhibitors (SNRIs) group (duloxetine, venlafaxine, milnacipran) and the atypical antidepressants group (bupropion, trazodone, mirtazapine, nefazodone). In this review, we present the available publications on TCAs (amitriptyline, doxepin, imipramine, desipramine, nortriptyline), TeCAs (amoxapine, maprotiline), selective serotonin reuptake inhibitors (SSRIs) (citalopram, fluoxetine, paroxetine), SNRIs (duloxetine, venlafaxine, milnacipran) and atypical antidepressants (bupropion) for the treatment of neuropathic pain. We also review analgesics acting as both opioid receptor agonists and also acting as aminergic reuptake inhibitors. Existing data are insufficient to conclude which of these new classes of antidepressants has the best clinical profile and will be the most effective in the treatment of neuropathic pain; in addition, a lower incidence of side effects should be considered. Increased experimental and translational research is a key for further improvement of the treatment of chronic pain with antidepressants. However

  1. Study of Synthetic Vision Systems (SVS) and Velocity-vector Based Command Augmentation System (V-CAS) on Pilot Performance

    NASA Technical Reports Server (NTRS)

    Liu, Dahai; Goodrich, Ken; Peak, Bob

    2006-01-01

    This study investigated the effects of synthetic vision system (SVS) concepts and advanced flight controls on single pilot performance (SPP). Specifically, we evaluated the benefits and interactions of two levels of terrain portrayal, guidance symbology, and control-system response type on SPP in the context of lower-landing minima (LLM) approaches. Performance measures consisted of flight technical error (FTE) and pilot perceived workload. In this study, pilot rating, control type, and guidance symbology were not found to significantly affect FTE or workload. It is likely that transfer from prior experience, limited scope of the evaluation task, specific implementation limitations, and limited sample size were major factors in obtaining these results.

  2. Closure Report for Corrective Action Unit 398: Area 25 Spill Sites, Nevada Test Site, Nevada

    SciTech Connect

    K. B. Campbell

    2003-04-01

    This Closure Report (CR) documents the activities performed to close Corrective Action Unit (CAU) 398: Area 25 Spill Sites, in accordance with the Federal Facility Agreement and Consent Order (FFACO) of 1996, and the Nevada Division of Environmental Protection (NDEP)-approved Streamlined Approach for Environmental Restoration (SA4FER) Plan for CAU 398: Area 25 Spill Sites, Nevada Test Site, Nevada (U.S. Department of Energy, Nevada Operations Office [DOEN], 2001). CAU 398 consists of the following thirteen Corrective Action Sites (CASs) all located in Area 25 of the Nevada Test Site (NTS) (Figure 1): CAS 25-25-02, Oil Spills, CAS 25-25-03, Oil Spills, CAS 25-25-04, Oil Spills, CAS 25-25-05, Oil Spills, CAS 25-25-06, Oil Spills, CAS 25-25-07, Hydraulic Oil Spill(s), CAS 25-25-08, Hydraulic Oil Spill(s), CAS 25-25-16, Diesel Spill (from CAS 25-01-02), CAS 25-25-17, Subsurface Hydraulic Oil Spill, CAS 25-44-0 1, Fuel Spill, CAS 25-44-04, Acid Spill (from CAS 25-01-01), CAS 25-44-02, Spill, and CAS 25-44-03, Spill. Copies of the analytical results for the site verification samples are included in Appendix B. Copies of the CAU Use Restriction Information forms are included in Appendix C.

  3. Closure Report for Corrective Action Unit 346: Areas 8, 10 Housekeeping Sites, Nevada Test Site, Nevada

    SciTech Connect

    K. B. Campbell

    2003-08-01

    This Closure Report documents the closure activities conducted for Corrective Action Unit (CAU) 346: Areas 8, 10 Housekeeping Sites. CAU 346 is listed in Appendix III of the Federal Facility Agreement and Consent Order (FFACO, 1996) and consists of the following 14 Corrective Action Sites (CASs) located in Areas 8 and 10 of the Nevada Test Site (NTS): (1) CAS 08-22-04: Drums (2); (2) CAS 08-22-11: Drums; Bucket; (3) CAS 08-24-02: Battery; (4) CAS 10-14-01: Transformer; (5) CAS 10-22-06: Drum (Gas Block); (6) CAS 10-22-10: Drum (Gas Block); (7) CAS 10-22-12: Drum (Gas Block); (8) CAS 10-22-13: Drum (Gas Block); (9) CAS 10-22-16: Drum (Gas Block); (10) CAS 10-22-22: Drum; (11) CAS 10-22-25: Drum; (12) CAS 10-22-36: Paint Can; (13) CAS 10-22-37: Gas Block; and (14) CAS 10-24-11: Battery. Closure activities consisted of closing each CAS by removing debris and/or material, disposing of the generated waste, and verifying that each site was clean-closed by visual inspection and/or laboratory analysis of soil verification samples.

  4. Observational Study Of The Pacific Western Boundary Currents And The Indonesian Throughflow by the CAS Strategic Priority Project

    NASA Astrophysics Data System (ADS)

    Yuan, D.; Wang, J.

    2014-12-01

    The warm pool in the western Pacific Ocean has significant impact on the evolution of ENSO and the East Asian monsoon. Ocean circulation in the western Pacific Ocean and in Indonesian seas plays an important role in the interannual climate variations and predictability of the tropical Indo-Pacific Ocean. A major observational program of the Chinese Academy of Sciences is recently launched to study the western Pacific Ocean circulation and the warm pool to test these scientific hypotheses. The physical oceanography project called the "Western Pacific Ocean Circulation and the Warm Pool Variability" is by far the largest and the most intensive observational program in history in the western Pacific ocean study. In this talk, the background and scientific hypotheses of the project, the observational design in the western Pacific Ocean, Indonesian seas, and the eastern Indian Ocean region, and some preliminary results of the program will be presented. The talk serves to encourage more scientists to collaborate in the studies of the ocean circulation and climate in the western Pacific and eastern Indian Oceans.

  5. A Cloud-Resolving Modeling Intercomparison Study on Properties of Cloud Microphysics, Convection, and Precipitation for a Squall Line Cas

    NASA Astrophysics Data System (ADS)

    Fan, J.; Han, B.; Morrison, H.; Varble, A.; Mansell, E.; Milbrandt, J.; Wang, Y.; Lin, Y.; Dong, X.; Giangrande, S. E.; Jensen, M. P.; Collis, S. M.; North, K.; Kollias, P.

    2015-12-01

    The large spread in CRM model simulations of deep convection and aerosol effects on deep convective clouds (DCCs) makes it difficult (1) to further our understanding of deep convection and (2) to define "benchmarks" and recommendations for their use in parameterization developments. Past model intercomparison studies used different models with different complexities of dynamic-microphysics interactions, making it hard to isolate the causes of differences between simulations. In this intercomparison study, we employed a much more constrained approach - with the same model and same experiment setups for simulations with different cloud microphysics schemes (one-moment, two-moment, and bin models). Both the piggybacking and interactive approaches are employed to explore the major microphysical processes that control the model differences and the significance of their feedback to dynamics through latent heating/cooling and cold pool characteristics. Real-case simulations are conducted for the squall line case 20 May 2011 from the MC3E field campaign. Results from the piggybacking approach show substantially different responses of the microphysics schemes to the same dynamical fields. Although the interactive microphysics-dynamics simulations buffer some differences compared with those from the piggyback runs, large differences still exist and are mainly contributed by ice microphysical processes parameterizations. The presentation will include in-depth analyses of the major microphysical processes for the squall line case, the significance of the feedback of the processes to dynamics, and how those results differ in different cloud microphysics schemes.

  6. Exploiting CRISPR/Cas systems for biotechnology

    PubMed Central

    Sampson, Timothy R.; Weiss, David S.

    2015-01-01

    The Cas9 endonuclease is the central component of the Type II CRISPR/Cas system, a prokaryotic adaptive restriction system against invading nucleic acids, such as those originating from bacteriophages and plasmids. Recently, this RNA-directed DNA endonuclease has been harnessed to target DNA sequences of interest. Here, we review the development of Cas9 as an important tool to not only edit the genomes of a number of different prokaryotic and eukaryotic species, but also as an efficient system for site-specific transcriptional repression or activation. Additionally, a specific Cas9 protein has been observed to target an RNA substrate, suggesting that Cas9 may have the ability to be programmed to target RNA as well. Cas proteins from other CRISPR/Cas subtypes may also be exploited in this regard. Thus, CRISPR/Cas systems represent an effective and versatile biotechnological tool, which will have significant impact on future advancements in genome engineering. PMID:24323919

  7. BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130Cas

    PubMed Central

    Borre, Pierre Vanden; Near, Richard I.; Makkinje, Anthony; Mostoslavsky, Gustavo; Lerner, Adam

    2011-01-01

    BCAR3 binds to the carboxy-terminus of p130Cas, a focal adhesion adapter protein. Both BCAR3 and p130Cas have been linked to resistance to anti-estrogens in breast cancer, Rac activation and cell motility. Using R743A BCAR3, a point mutant that has lost the ability to bind p130Cas, we find that BCAR3-p130Cas complex formation is not required for BCAR3-mediated anti-estrogen resistance, Rac activation or discohesion of epithelial breast cancer cells. Complex formation was also not required for BCAR3-induced lamellipodia formation in BALB/c-3T3 fibroblasts but was required for optimal BCAR3-induced motility. Although both wildtype and R743A BCAR3 induced phosphorylation of p130Cas and the related adapter protein HEF1/NEDD9, chimeric NSP3:BCAR3 experiments demonstrate that such phosphorylation does not correlate with BCAR3-induced anti-estrogen resistance or lamellipodia formation. Wildtype but not R743A BCAR3 induced lamellipodia formation and augmented cell motility in p130Cas-/- murine embryonic fibroblasts (MEFs), suggesting that while p130Cas itself is not strictly required for these endpoints, complex formation with other CAS family members is, at least in cells lacking p130Cas. Overall, our work suggests that many, but not all, BCAR3-mediated signaling events in epithelial and mesenchymal cells are independent of p130Cas association. These studies also indicate that disruption of the BCAR3-p130Cas complex is unlikely to reverse BCAR3-mediated anti-estrogen resistance. PMID:21262352

  8. Studies on the Mechanism of Action of Dinitramine

    PubMed Central

    Travis, Robert L.; Woods, William G.

    1977-01-01

    The effect of dinitramine, a selective herbicide, on the plasma membrane of the soybean (Glycine max L.) root was studied. Used as marker systems to observe the herbicide effect were two plasma-membrane-specific enzymes, pH 6.5 ATPase and glucan synthetase. The activity of pH 6.5 ATPase decreased significantly in membrane vesicles prepared from roots harvested 15 minutes after treatment with dinitramine. Maximum inhibition occurred in roots harvested 2 hours after treatment. Glucan synthetase activity decreased similarly within 2 hours of treatment. Membrane permeability to 86Rb was rapidly increased by dinitramine. The activity of pH 6.5 ATPase returned to the control level within 8 hours of treatment with dinitramine. These results show dinitramine's initial site of action to be the plasma membrane, producing an over-all reduction in membrane function through inactivation of membrane-associated proteins. PMID:16660043

  9. Prospects and challenges of CRISPR/Cas genome editing for the study and control of neglected vector-borne nematode diseases.

    PubMed

    Zamanian, Mostafa; Andersen, Erik C

    2016-09-01

    Neglected tropical diseases caused by parasitic nematodes inflict an immense health and socioeconomic burden throughout much of the developing world. Current estimates indicate that more than two billion people are infected with nematodes, resulting in the loss of 14 million disability-adjusted life years per annum. Although these parasites cause significant mortality, they primarily cause chronic morbidity through a wide range of severe clinical ailments. Treatment options for nematode infections are restricted to a small number of anthelmintic drugs, and the rapid expansion of anthelmintic mass drug administration raises concerns of drug resistance. Preservation of existing drugs is necessary, as well as the development of new treatment options and methods of control. We focus this review on how the democratization of CRISPR/Cas9 genome editing technology can be enlisted to improve our understanding of the biology of nematode parasites and our ability to treat the infections they cause. We will first explore how this robust method of genome manipulation can be used to newly exploit the powerful model nematode Caenorhabditis elegans for parasitology research. We will then discuss potential avenues to develop CRISPR/Cas9 editing protocols in filarial nematodes. Lastly, we will propose potential ways in which CRISPR/Cas9 can be used to engineer gene drives that target the transmission of mosquito-borne filarial nematodes. PMID:27300487

  10. 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…

  11. "CAS" Statement of Shared Ethical Principles

    ERIC Educational Resources Information Center

    Council for the Advancement of Standards in Higher Education, 2006

    2006-01-01

    The Council for the Advancement of Standards in Higher Education (CAS) has served as a voice for quality assurance and promulgation of standards in higher education for over 25 years. CAS was established to promote inter-association efforts to address quality assurance, student learning, and professional integrity. CAS includes membership of over…

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

    PubMed

    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. How Action and Context Priming Influence Categorization: A Developmental Study

    ERIC Educational Resources Information Center

    Kalenine, Solene; Bonthoux, Francoise; Borghi, Anna M.

    2009-01-01

    Embodied views of cognition propose that concepts are grounded in sensorimotor experience. Diverse aspects of sensorimotor experience, like action and context information, could play a key role in the formation and processing of manipulable object concepts. Specifically, contextual information could help to link specific actions experienced with…

  14. Insight and Action Analytics: Three Case Studies to Consider

    ERIC Educational Resources Information Center

    Milliron, Mark David; Malcolm, Laura; Kil, David

    2014-01-01

    Civitas Learning was conceived as a community of practice, bringing together forward-thinking leaders from diverse higher education institutions to leverage insight and action analytics in their ongoing efforts to help students learn well and finish strong. We define insight and action analytics as drawing, federating, and analyzing data from…

  15. CORRECTIVE ACTION PLAN FOR CORRECTIVE ACTION UNIT 528: POLYCHLORINATED BIPHENYLS CONTAMINATION NEVADA TEST SITE, NEVADA

    SciTech Connect

    BECHTEL NEVADA

    2005-06-01

    Corrective Action Unit (CAU) 528: Polychlorinated Biphenyls Contamination is listed in Appendix III of the Federal Facility Agreement and Consent Order (FFACO, 1996) and is located in Area 25 of the Nevada Test Site. CAU 528 was created to address polychlorinated biphenyl (PCB) contamination identified during the CAU 262 corrective action investigation. CAU 528 consists of one Corrective Action Site (CAS): CAS 25-27-03, Polychlorinated Biphenyls Surface Contamination.

  16. A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion

    PubMed Central

    Sampson, Timothy R.; Napier, Brooke A.; Schroeder, Max R.; Louwen, Rogier; Zhao, Jinshi; Chin, Chui-Yoke; Ratner, Hannah K.; Llewellyn, Anna C.; Jones, Crystal L.; Laroui, Hamed; Merlin, Didier; Zhou, Pei; Endtz, Hubert P.; Weiss, David S.

    2014-01-01

    Clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals. PMID:25024199

  17. Nitazoxanide: Nematicidal Mode of Action and Drug Combination Studies

    PubMed Central

    Somvanshi, Vishal S.; Ellis, Brian L.; Hu, Yan; Aroian, Raffi V.

    2014-01-01

    Intestinal nematodes or roundworms (aka soil-transmitted helminths or STHs) cause great disease. They infect upwards of two billion people, leading to high morbidity and a range of health problems, especially in infected children and pregnant women. Development of resistance to the two main classes of drugs used to treat intestinal nematode infections of humans has been reported. To fight STH infections, we need new and more effective drugs and ways to improve the efficacy of the old drugs. One promising alternative drug is nitazoxanide (NTZ). NTZ, approved for treating human protozoan infections, was serendipitously shown to have therapeutic activity against STHs. However, its mechanism of action against nematodes is not known. Using the laboratory nematode Caenorhabditis elegans, we show that NTZ acts on the nematodes through avr-14, an alpha-type subunit of a glutamate-gated chloride ion channel known for its role in ivermectin susceptibility. In addition, a forward genetic screen to select C. elegans mutants resistant to NTZ resulted in isolation of two NTZ resistant mutants that are not in avr-14, suggesting that additional mechanisms are involved in resistance to NTZ. We found that NTZ combines synergistically with other classes of anthelmintic drugs, i.e. albendazole and pyrantel, making it a good candidate for further studies on its use in drug combination therapy of STH infections. Given NTZ acts against a wide range of nematode parasites, our findings also validate avr-14 as an excellent target for pan-STH therapy. PMID:24412397

  18. Mode of Action Studies on Nitrodiphenyl Ether Herbicides

    PubMed Central

    Bowyer, John R.; Smith, Beverly J.; Camilleri, Patrick; Lee, Susan A.

    1987-01-01

    5-[2-Chloro-4-(trifluoromethyl)phenoxy]-2-nitroacetophenone oxime-o-(acetic acid, methyl ester) (DPEI), is a potent nitrodiphenyl ether herbicide which causes rapid leaf wilting, membrane lipid peroxidation, and chlorophyll destruction in a process which is both light- and O2-dependent. These effects resemble those of other nitrodiphenyl ether herbicides. Unlike paraquat, the herbicidal effects of DPEI are only slightly reduced by pretreatment with the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. DPEI is a weak inhibitor of photosynthetic electron transport (I50 15 micromolar for water to paraquat) in vitro, with at least one site of action at the cytochrome b6f complex. Ultrastructural studies and measurements of ethane formation resulting from lipid peroxidation indicate that mutants of barley lacking photosystem I (PSI) (viridis-zb63) or photosystem II (viridis-zd69) are resistant to paraquat but susceptible to DPEI. The results indicate that electron transfer through both photosystems is not essential for the toxic effects of nitrodiphenyl ether herbicides. Furthermore, the results show that neither cyclic electron transport around PSI, nor the diversion of electrons from PSI to O2 when NADPH consumption is blocked are essential for the phytotoxicity of nitrodiphenyl ether herbicides. Images Fig. 2 Fig. 3 Fig. 4 PMID:16665297

  19. Melatonin action in tumor skeletal muscle cells: an ultrastructural study.

    PubMed

    Burattini, S; Battistelli, M; Codenotti, S; Falcieri, E; Fanzani, A; Salucci, S

    2016-04-01

    Melatonin (Mel), or N-acetyl-5-methoxytryptamine, is a circadian hormone that can diffuse through all the biological membranes thanks to its amphiphilic structure, also overcoming the blood-brain barrier and placenta. Although Mel has been reported to exhibit strong antioxidant properties in healthy tissues, studies carried out on tumor cultures gave a different picture of its action, often describing Mel as effective to trigger the cell death of tumor cells by enhancing oxidative stress. Based on this premise, here Mel effect was investigated using a tumor cell line representative of the human alveolar rhabdomyosarcoma (ARMS), the most frequent soft tissue sarcoma affecting childhood. For this purpose, Mel was given either dissolved in ethanol (EtOH) or dimethyl sulfoxide (DMSO) at different concentrations and time exposures. Cell viability assays and ultrastructural observations demonstrated that Mel was able to induce a dose- and time-dependent cell death independently on the dissolution solvent. Microscopy analyses highlighted the presence of various apoptotic and necrotic patterns correlating with the increasing Mel dose and time of exposure. These findings suggest that Mel, triggering apoptosis in ARMS cells, could be considered as a promising drug for future multitargeted therapies. PMID:26953151

  20. Exploring Coaching Actions Based on Developed Values: A Case Study of a Female Hockey Coach

    ERIC Educational Resources Information Center

    Callary, Bettina; Werthner, Penny; Trudel, Pierre

    2013-01-01

    There are few empirical studies that demonstrate how values are developed and how they are linked to coaching actions. There can be a discrepancy between the statement of coaches' values and their actual coaching actions. In order to examine how coaching actions are influenced by values that are developed over a lifetime, the purpose of this…

  1. Computational Neural Modeling of Speech Motor Control in Childhood Apraxia of Speech (CAS)

    ERIC Educational Resources Information Center

    Terband, Hayo; Maassen, Ben; Guenther, Frank H.; Brumberg, Jonathan

    2009-01-01

    Purpose: Childhood apraxia of speech (CAS) has been associated with a wide variety of diagnostic descriptions and has been shown to involve different symptoms during successive stages of development. In the present study, the authors attempted to associate the symptoms of CAS in a particular developmental stage with particular…

  2. Phonological Awareness and Early Reading Development in Childhood Apraxia of Speech (CAS)

    ERIC Educational Resources Information Center

    McNeill, B. C.; Gillon, G. T.; Dodd, B.

    2009-01-01

    Background: Childhood apraxia of speech (CAS) is associated with phonological awareness, reading, and spelling deficits. Comparing literacy skills in CAS with other developmental speech disorders is critical for understanding the complexity of the disorder. Aims: This study compared the phonological awareness and reading development of children…

  3. DNA fragment editing of genomes by CRISPR/Cas9.

    PubMed

    Jinhuan, Li; Jia, Shou; Qiang, Wu

    2015-10-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system from bacteria and archaea emerged recently as a new powerful technology of genome editing in virtually any organism. Due to its simplicity and cost effectiveness, a revolutionary change of genetics has occurred. Here, we summarize the recent development of DNA fragment editing methods by CRISPR/Cas9 and describe targeted DNA fragment deletions, inversions, duplications, insertions, and translocations. The efficient method of DNA fragment editing provides a powerful tool for studying gene function, regulatory elements, tissue development, and disease progression. Finally, we discuss the prospects of CRISPR/Cas9 system and the potential applications of other types of CRISPR system. PMID:26496751

  4. Action of Molecular Switches in GPCRs - Theoretical and Experimental Studies

    PubMed Central

    Trzaskowski, B; Latek, D; Yuan, S; Ghoshdastider, U; Debinski, A; Filipek, S

    2012-01-01

    G protein coupled receptors (GPCRs), also called 7TM receptors, form a huge superfamily of membrane proteins that, upon activation by extracellular agonists, pass the signal to the cell interior. Ligands can bind either to extracellular N-terminus and loops (e.g. glutamate receptors) or to the binding site within transmembrane helices (Rhodopsin-like family). They are all activated by agonists although a spontaneous auto-activation of an empty receptor can also be observed. Biochemical and crystallographic methods together with molecular dynamics simulations and other theoretical techniques provided models of the receptor activation based on the action of so-called “molecular switches” buried in the receptor structure. They are changed by agonists but also by inverse agonists evoking an ensemble of activation states leading toward different activation pathways. Switches discovered so far include the ionic lock switch, the 3-7 lock switch, the tyrosine toggle switch linked with the nPxxy motif in TM7, and the transmission switch. The latter one was proposed instead of the tryptophan rotamer toggle switch because no change of the rotamer was observed in structures of activated receptors. The global toggle switch suggested earlier consisting of a vertical rigid motion of TM6, seems also to be implausible based on the recent crystal structures of GPCRs with agonists. Theoretical and experimental methods (crystallography, NMR, specific spectroscopic methods like FRET/BRET but also single-molecule-force-spectroscopy) are currently used to study the effect of ligands on the receptor structure, location of stable structural segments/domains of GPCRs, and to answer the still open question on how ligands are binding: either via ensemble of conformational receptor states or rather via induced fit mechanisms. On the other hand the structural investigations of homo- and heterodimers and higher oligomers revealed the mechanism of allosteric signal transmission and receptor

  5. Difficult action decisions reduce the sense of agency: A study using the Eriksen flanker task.

    PubMed

    Sidarus, Nura; Haggard, Patrick

    2016-05-01

    The sense of agency refers to the feeling that we are in control of our actions and, through them, of events in the outside world. Much research has focused on the importance of retrospectively matching predicted and actual action outcomes for a strong sense of agency. Yet, recent studies have revealed that a metacognitive signal about the fluency of action selection can prospectively inform our sense of agency. Fluent, or easy, action selection leads to a stronger sense of agency over action outcomes than dysfluent, or difficult, selection. Since these studies used subliminal priming to manipulate action selection, it remained unclear whether supraliminal stimuli affecting action selection would have similar effects. We used supraliminal flankers to manipulate action selection in response to a central target. Experiment 1 revealed that conflict in action selection, induced by incongruent flankers and targets, led to reduced agency ratings over an outcome that followed the participant's response, relative to neutral and congruent flanking conditions. Experiment 2 replicated this result, and extended it to free choice between alternative actions. Finally, Experiment 3 varied the stimulus onset asynchrony (SOA) between flankers and target. Action selection performance varied with SOA. Agency ratings were always lower in incongruent than congruent trials, and this effect did not vary across SOAs. Sense of agency is influenced by a signal that tracks conflict in action selection, regardless of the visibility of stimuli inducing conflict, and even when the timing of the stimuli means that the conflict may not affect performance. PMID:27017411

  6. CAS-1 lunar soil simulant

    NASA Astrophysics Data System (ADS)

    Zheng, Yongchun; Wang, Shijie; Ouyang, Ziyuan; Zou, Yongliao; Liu, Jianzhong; Li, Chunlai; Li, Xiongyao; Feng, Junming

    2009-02-01

    Lunar soil simulant is a geochemical reproduction of lunar regolith, and is needed for lunar science and engineering researches. This paper describes a new lunar soil simulant, CAS-1, prepared by the Chinese Academy of Sciences, to support lunar orbiter, soft-landing mission and sample return missions of China’s Lunar Exploration Program, which is scheduled for 2004 2020. Such simulants should match the samples returned from the Moon, all collected from the lunar regolith rather than outcrops. The average mineral and chemical composition of lunar soil sample returned from the Apollo 14 mission, which landed on the Fra Mauro Formation, is chosen as the model for the CAS-1 simulant. Source material for this simulant was a low-Ti basaltic scoria dated at 1600 years from the late Quaternary volcanic area in the Changbai Mountains of northeast China. The main minerals of this rock are pyroxene, olivine, and minor plagioclase, and about 20 40% modal glass. The scoria was analyzed by XRF and found to be chemically similar to Apollo 14 lunar sample 14163. It was crushed in an impact mill with a resulting median particle size 85.9 μm, similar to Apollo soils. Bulk density, shear resistance, complex permittivity, and reflectance spectra were also similar to Apollo 14 soil. We conclude that CAS-1 is an ideal lunar soil simulant for science and engineering research of future lunar exploration program.

  7. Applications of CRISPR-Cas systems in neuroscience

    PubMed Central

    Heidenreich, Matthias; Zhang, Feng

    2016-01-01

    Genome editing tools, and in particular those based on CRISPR-Cas systems, are accelerating the pace of biological research and enabling targeted genetic interrogation in virtually any organism and cell type. These tools have opened the door to the development of new model systems for studying the complexity of the nervous system, including animal and stem cell-derived in vitro models. Precise and efficient gene editing using CRISPR-Cas systems has the potential to advance both basic and translational neuroscience research. PMID:26656253

  8. Actionable Postcolonial Theory in Education. Postcolonial Studies in Education

    ERIC Educational Resources Information Center

    Andreotti, Vanessa

    2011-01-01

    "Actionable Postcolonial Theory in Education" illustrates how postcolonial theory can be put to work in education. It offers an accessible and handy overview and comparison of postcolonial theory and other theoretical debates related to critiques of Western ethnocentrism and hegemony. It also offers examples that illustrate how a discursive strand…

  9. Action Research. Case Studies in TESOL Practice Series.

    ERIC Educational Resources Information Center

    Edge, Julian, Ed.

    Chapter titles in this book include the following: "Attitude and Access: Building a New Teaching/Learning Community in TESOL" (Julian Edge); "Here It Is, Rough Though It May Be: Basic Computer for ESL" (Alison Perkins); "An 'It's Not Action Research Yet, but I'm Getting There' Approach to Teaching Writing" (Neil Cowie); "Early Reflections:…

  10. Motivating Struggling Adolescent Readers: An Action Research Study

    ERIC Educational Resources Information Center

    Richardson, Eileen M.

    2016-01-01

    In a high-school reading class, the author used Reader's Theater as an instructional and motivational strategy for underachieving students. This action research focused on the extent to which implementing Reader's Theater motivated students to read and improve their reading skills. Consistent increases in scores for all students occurred over the…

  11. Comparative Actions of Barbiturates Studied by Pollen Grain Germination.

    ERIC Educational Resources Information Center

    Kordan, Herbert A.; Mumford, Pauline M.

    1979-01-01

    Describes a simple experimental system whereby the comparative actions of long, medium, and short-acting barbiturates can be demonstrated in a relatively short period of time under optical microscopy using pollen grains as the biological test or assay system. (Author/HM)

  12. Action Learning with Second Life--A Pilot Study

    ERIC Educational Resources Information Center

    Wagner, Christian; Ip, Rachael K. F.

    2009-01-01

    Virtual worlds, computer-based simulated environments in which users interact via avatars, provide an opportunity for the highly realistic enactment of real life activities online. Unlike computer games, which have a pre-defined purpose, pay-off structure, and action patterns, virtual worlds can leave many of these elements for users to determine.…

  13. Corrective Action Plan for Corrective Action Unit 139: Waste Disposal Sites, Nevada Test Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2007-07-01

    Corrective Action Unit (CAU) 139, Waste Disposal Sites, is listed in the Federal Facility Agreement and Consent Order (FFACO) of 1996 (FFACO, 1996). CAU 139 consists of seven Corrective Action Sites (CASs) located in Areas 3, 4, 6, and 9 of the Nevada Test Site (NTS), which is located approximately 65 miles (mi) northwest of Las Vegas, Nevada (Figure 1). CAU 139 consists of the following CASs: CAS 03-35-01, Burn Pit; CAS 04-08-02, Waste Disposal Site; CAS 04-99-01, Contaminated Surface Debris; CAS 06-19-02, Waste Disposal Site/Burn Pit; CAS 06-19-03, Waste Disposal Trenches; CAS 09-23-01, Area 9 Gravel Gertie; and CAS 09-34-01, Underground Detection Station. Details of the site history and site characterization results for CAU 139 are provided in the approved Corrective Action Investigation Plan (CAIP) (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2006) and in the approved Corrective Action Decision Document (CADD) (NNSA/NSO, 2007). The purpose of this Corrective Action Plan (CAP) is to present the detailed scope of work required to implement the recommended corrective actions as specified in Section 4.0 of the approved CADD (NNSA/NSO, 2007). The approved closure activities for CAU 139 include removal of soil and debris contaminated with plutonium (Pu)-239, excavation of geophysical anomalies, removal of surface debris, construction of an engineered soil cover, and implementation of use restrictions (URs). Table 1 presents a summary of CAS-specific closure activities and contaminants of concern (COCs). Specific details of the corrective actions to be performed at each CAS are presented in Section 2.0 of this report.

  14. Corrective Action Plan for Corrective Action Unit 543: Liquid Disposal Units, Nevada Test Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2006-09-01

    Corrective Action Unit (CAU) 543, Liquid Disposal Units, is listed in Appendix III of the Federal Facility Agreement and Consent Order of 1996. CAU 543 consists of seven Corrective Action Sites (CASs) located in Areas 6 and 15 of the Nevada Test Site, which is approximately 65 miles northwest of Las Vegas, Nevada. CAU 543 consists of the following seven CASs: {sm_bullet} CAS 06-07-01, Decon Pad {sm_bullet} CAS 15-01-03, Aboveground Storage Tank {sm_bullet} CAS 15-04-01, Septic Tank {sm_bullet} CAS 15-05-01, Leachfield {sm_bullet} CAS 15-08-01, Liquid Manure Tank {sm_bullet} CAS 15-23-01, Underground Radioactive Material Area {sm_bullet} CAS 15-23-03, Contaminated Sump, Piping From January 24, 2005 through April 14, 2005, CAU 543 site characterization activities were conducted, and are reported in Appendix A of the CAU 543 Corrective Action Decision Document (CADD) (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2005). The recommended corrective action as stated in the approved CADD is No Further Action for five of the CAU 543 CASs, and Closure In Place for the remaining two CASs.

  15. Evaluation of a peripherally selective antiandrogen (Casodex) as a tool for studying the relationship between testosterone and spermatogenesis in the rat.

    PubMed

    Chandolia, R K; Weinbauer, G F; Behre, H M; Nieschlag, E

    1991-03-01

    The endocrine profile and the effects on spermatogenesis of the new antiandrogen, Casodex [2RS)-4-cyano-3-(4-fluorophenylsulphonyl)-2-anilide, CAS) were evaluated in the adult rat. In the first experiment rats were administered CAS at daily doses of 10, 20 and 40 mg/kg for 14 days. For comparison groups receiving flutamide (FL, 10 mg/kg) and ethane dimethane sulphonate (EDS) were included. Unlike FL, administration of CAS (10 and 20 mg/kg) did not significantly raise serum concentrations of gonadotropic hormones and testosterone. With 40 mg/kg CAS gonadotropin secretion, but not testosterone levels, were elevated on day 15. Administration of CAS lowered the weight of the seminal vesicles and coagulating glands comparable to the administration of the Leydig cell toxin EDS. In contrast to FL a significant loss of germ cells in stage VII of spermatogenesis was observed with CAS. In a second experiment the ability of FL and CAS to block testicular androgen action was compared in rats with reduced testicular androgen production induced by a gonadotropin-releasing hormone antagonist. Both antiandrogens markedly enhanced spermatogenic involution as revealed by quantitative flow cytometric analysis of germ cell numbers. The study demonstrates that (a) CAS is a peripherally selective antiandrogen and (b) CAS might provide a feasible approach to study androgen dependence of spermatogenesis in the presence of normal FSH levels. PMID:1848993

  16. Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9.

    PubMed

    Anders, Carolin; Bargsten, Katja; Jinek, Martin

    2016-03-17

    The RNA-guided endonuclease Cas9 from Streptococcus pyogenes (SpCas9) forms the core of a powerful genome editing technology. DNA cleavage by SpCas9 is dependent on the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) in the target DNA, restricting the choice of targetable sequences. To address this limitation, artificial SpCas9 variants with altered PAM specificities have recently been developed. Here we report crystal structures of the VQR, EQR, and VRER SpCas9 variants bound to target DNAs containing their preferred PAM sequences. The structures reveal that the non-canonical PAMs are recognized by an induced fit mechanism. Besides mediating sequence-specific base recognition, the amino acid substitutions introduced in the SpCas9 variants facilitate conformational remodeling of the PAM region of the bound DNA. Guided by the structural data, we engineered a SpCas9 variant that specifically recognizes NAAG PAMs. Taken together, these studies inform further development of Cas9-based genome editing tools. PMID:26990992

  17. Breast Cancer Anti-estrogen Resistance 3 (BCAR3) Protein Augments Binding of the c-Src SH3 Domain to Crk-associated Substrate (p130cas)*

    PubMed Central

    Makkinje, Anthony; Vanden Borre, Pierre; Near, Richard I.; Patel, Prayag S.; Lerner, Adam

    2012-01-01

    The focal adhesion adapter protein p130cas regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130cas. AND-34/BCAR3, one of three NSP family members, binds the p130cas carboxyl terminus, adjacent to a bipartite p130cas Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130cas. Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130cas complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130cas to bind the Src SH3 domain through an RPLPSPP motif in the p130cas SBD. Although our prior work identified phosphorylation of the serine within the p130cas RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130cas. The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130cas complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130cas substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130cas. Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130cas and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130cas complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130cas SBD. PMID:22711540

  18. Targeting cellular mRNAs translation by CRISPR-Cas9

    PubMed Central

    Liu, Yuchen; Chen, Zhicong; He, Anbang; Zhan, Yonghao; Li, Jianfa; Liu, Li; Wu, Hanwei; Zhuang, Chengle; Lin, Junhao; Zhang, Qiaoxia; Huang, Weiren

    2016-01-01

    Recently CRISPR-Cas9 system has been reported to be capable of targeting a viral RNA, and this phenomenon thus raises an interesting question of whether Cas9 can also influence translation of cellular mRNAs. Here, we show that both natural and catalytically dead Cas9 can repress mRNA translation of cellular genes, and that only the first 14 nt in the 5′ end of sgRNA is essential for this process. CRISPR-Cas9 can suppress the protein expression of an unintended target gene without affecting its DNA sequence and causes unexpected phenotypic changes. Using the designed RNA aptamer-ligand complexes which physically obstruct translation machinery, we indicate that roadblock mechanism is responsible for this phenomenon. Our work suggests that studies on Cas9 should avoid the potential off-target effects by detecting the alteration of genes at both the DNA and protein levels. PMID:27405721

  19. Complete active space (CAS) SCF study of the dipole polarizability function for the X 1Sigma + state of LiH

    NASA Astrophysics Data System (ADS)

    Roos, Björn O.; Sadlej, Andrzej J.

    1982-06-01

    The CAS SCF method is applied to the calculation of the dipole polarizability function of the X 1Σ+ state of LiH by using the finite-field perturbation approach. The dipole moment function and the potential energy curve are also computed. The vibrational averaging of electric properties has been carried out with different accurate potential energy curves available for the LiH molecule. It has been found that the vibrational contribution to the polarizability components leads to the change of the sign of the polarizability anisotropy between the second and third vibrational state. The dipole polarizability tensor transition matrix elements for some of the lowest energy vibrational transitions have been calculated and can be checked against the Raman intensity data.

  20. Studies of the potential genotoxic effects of furoxans: the case of CAS 1609 and of the water-soluble analogue of CHF 2363.

    PubMed

    Balbo, S; Lazzarato, L; Di Stilo, A; Fruttero, R; Lombaert, N; Kirsch-Volders, M

    2008-04-21

    CAS 1609 (compound 1) and CHF 2363 (compound 2) are two furoxan derivatives able to release nitric oxide (NO) under physiological conditions, and display typical NO-dependent vasodilator activity. The potential genotoxic effects of compound 1 and of the water-soluble analogue of CHF 2363 (compound 2a) were investigated. The results show that the two compounds induce genotoxic effects only at concentrations that significantly reduce cell viability. However, in the case of compound 1 this range of concentrations is one order of magnitude higher than the one leading to the beneficial effects, while in the case of compound 2a these ranges partially overlap. In both cases the release of NO plays a key role in the induction of the cytotoxic and genotoxic effects, since the non-NO-donating furazan analogues display a different toxicological profile, and since the effects were reduced in the presence of oxyhaemoglobin, a well-known NO-scavenger. PMID:18378101

  1. Learning from Action Evaluation of the Use of Multimedia Case Studies in Management Information Systems Courses

    ERIC Educational Resources Information Center

    Kawulich, Barbara B.

    2011-01-01

    This manuscript shares lessons learned from conducting an action evaluation of the use of multimedia case studies in Management Information Systems (MIS) courses. Three undergraduate MIS classes took part in the study. The purpose for using case studies in these classes was to teach students about the role of MIS in business. An action evaluation…

  2. Prospective Mathematics Teachers' Interactions with CAS-Based Textbook Elements

    ERIC Educational Resources Information Center

    Davis, Jon D.

    2015-01-01

    This study investigated how a group of 10 prospective secondary mathematics teachers (PST) read, evaluated, and adapted a textbook lesson involving the symbolic manipulation capabilities of computer algebra systems (CASS). PST read the entire lesson and tended to focus on the organizing question at the beginning of the student lesson and the CAS-S…

  3. Creative Expression as a Way of Knowing in Diabetes Adult Health Education: An Action Research Study

    ERIC Educational Resources Information Center

    Stuckey, Heather L.

    2009-01-01

    This action research study explores the meaning-making process using forms of creative expression for eight women with insulin-dependent diabetes. The study is theoretically informed by arts-based ways of knowing and aspects of feminist poststructuralism, and explains the process of creativity used in the action research process. The findings…

  4. Developmental Advising for Marginalized Community College Students: An Action Research Study

    ERIC Educational Resources Information Center

    Jones, Terrica S.

    2013-01-01

    The purpose of this action research study was to understand, evaluate, and improve the developmental advising practices used at a Washington State community college. This action research study endeavored to strengthen the developmental advising model originally designed to support the college's marginalized students. Guiding questions for the…

  5. A School Action Plan with Stakeholder Involvement: A Case Study of One Elementary School

    ERIC Educational Resources Information Center

    Getty, Jacob J., Jr.

    2011-01-01

    This case study focused on a school action plan, using a planning and implementation process that focused on improving stakeholder involvement and responsibility for student reading achievement at Eisenberg Elementary School. This study examined the impact of the school action process on the development of a new plan compared to other traditional…

  6. The Impact of Special Education on Self-Perception: An Autoethnographic-Action Research Study

    ERIC Educational Resources Information Center

    Zabonick, Lisa A.

    2013-01-01

    This qualitative autoethnographic-action research study examined how lack of voice as a special education student in the mid-1970s influenced my self-perception. This study also examined, through the use of action research, what influence storytelling had on teacher perceptions of students with disabilities. Autoethnographic data results were used…

  7. Transforming Language Ideologies through Action Research: A Case Study of Bilingual Science Learning

    ERIC Educational Resources Information Center

    Yang, Eunah

    2012-01-01

    This qualitative case study explored a third grade bilingual teacher's transformative language ideologies through participating in a collaborative action research project. By merging language ideologies theory, Cultural Historical Activity Theory (CHAT), and action research, I was able to identify the analytic focus of this study. I analyzed…

  8. Experimental study of cyclic action of plasma on tungsten

    NASA Astrophysics Data System (ADS)

    Voronin, A. V.; Aleksandrov, A. E.; Ber, B. Ya.; Brunkov, P. N.; Bormatov, A. A.; Gusev, V. K.; Demina, E. V.; Novokhatskii, A. N.; Pavlov, S. I.; Prusakova, M. D.; Sotnikova, G. Yu.; Yagovkina, M. A.

    2016-03-01

    We report on experimental results on multiple action of hydrogen, deuterium, and helium plasmas produced by a plasma gun and the Globus-M tokamak on tungsten. The surface temperature in the course of irradiation is measured with a bichromatic pyrometer with a time resolution of ⩾1 μs. The morphology of the surface layer is investigated and X-ray structure analysis of tungsten exposed to multiple radiations by the plasma under various conditions is carried out. A slight decrease in the lattice parameter in the sample subjected to the maximal number of irradiation cycles is detected. It is shown that the morphology of the tungsten surface irradiated by the hydrogen plasma from the gun and by the deuterium plasma from the Globus-M tokamak changes (the structure becomes smoother). The characteristic depth of the layer in which impurities have been accumulated exceeds 0.5 μm. This depth was the largest for the sample exposed to 1000 shots from the gun and 2370 shots from the tokamak. It is shown that the helium jet from the plasma gun makes it possible to simulate the action of helium ions on the International Thermonuclear Experimental Reactor (ITER) diverter, producing a layer of submicrometer particles (bubbles).

  9. 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.

  10. p130Cas Scaffolds the Signalosome To Direct Adaptor-Effector Cross Talk during Kaposi's Sarcoma-Associated Herpesvirus Trafficking in Human Microvascular Dermal Endothelial Cells

    PubMed Central

    Bandyopadhyay, Chirosree; Veettil, Mohanan Valiya; Dutta, Sujoy

    2014-01-01

    , without any intrinsic enzymatic activity, are well known to allow a great diversity of specific and coordinated protein-protein interactions imparting signal amplification to different networks for physiological and pathological signaling. They are involved in integrating signals from growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. The present study identifies human microvascular dermal endothelial (HMVEC-d) cellular scaffold protein p130Cas (Crk-associated substrate) as a platform to promote Kaposi's sarcoma-associated herpesvirus (KSHV) trafficking. Early during KSHV de novo infection, p130Cas associates with lipid rafts and scaffolds EphrinA2 (EphA2)-associated critical adaptor members to downstream effector molecules, promoting successful nuclear delivery of the KSHV genome. Hence, simultaneous targeting of the receptor EphA2 and scaffolding action of p130Cas can potentially uncouple the signal cross talk of the KSHV entry-associated upstream signal complex from the immediate downstream trafficking-associated signalosome, consequently routing KSHV toward lysosomal degradation and eventually blocking KSHV infection and associated malignancies. PMID:25253349

  11. 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)

  12. The effect of Mycobacterium tuberculosis CRISPR-associated Cas2 (Rv2816c) on stress response genes expression, morphology and macrophage survival of Mycobacterium smegmatis.

    PubMed

    Huang, Qinqin; Luo, Hongping; Liu, Minqiang; Zeng, Jie; Abdalla, Abualgasim Elgaili; Duan, Xiangke; Li, Qiming; Xie, Jianping

    2016-06-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) are present in the genome of 40% bacteria and 90% archaea. CRISPR and accompanying Cas proteins constitute an adaptive immune system against disruptive mobile genetic elements. Two CRISPRs and 9 genes encoding CRISPR-associated proteins have been found in the genome of Mycobacterium tuberculosis. The CRISPR-associated Cas2 is an endoribonuclease required for the acquisition of new spacers. In this study, Cas2 encoded by Rv2816c was expressed in Mycobacterium smegmatis lacking CRISPR-Cas system and its role in stress responses of M. smegmatis in vitro and within macrophages was studied. We found that Cas2 mediated M. smegmatis stress response changes were associated with the altered expression of sigma factors which involved in mycobacterial stress response and virulence. We also found that Cas2 decreased the survival of M. smegmatis within macrophages. This study provides new insights on the role of Cas2. PMID:26498723

  13. Applying CRISPR-Cas9 tools to identify and characterize transcriptional enhancers.

    PubMed

    Lopes, Rui; Korkmaz, Gozde; Agami, Reuven

    2016-09-01

    The development of the CRISPR-Cas9 system triggered a revolution in the field of genome engineering. Initially, the use of this system was focused on the study of protein-coding genes but, recently, a number of CRISPR-Cas9-based tools have been developed to study non-coding transcriptional regulatory elements. These technological advances offer unprecedented opportunities for elucidating the functions of enhancers in their endogenous context. Here, we discuss the application, current limitations and future development of CRISPR-Cas9 systems to identify and characterize enhancer elements in a high-throughput manner. PMID:27381243

  14. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

    PubMed

    Burger, Alexa; Lindsay, Helen; Felker, Anastasia; Hess, Christopher; Anders, Carolin; Chiavacci, Elena; Zaugg, Jonas; Weber, Lukas M; Catena, Raul; Jinek, Martin; Robinson, Mark D; Mosimann, Christian

    2016-06-01

    CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo. PMID:27130213

  15. 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.

  16. Streamlined Approach for Environmental Restoration Plan for Corrective Action Unit 107: Low Impact Soil Sites, Nevada Test Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2008-09-30

    This Streamlined Approach for Environmental Restoration Plan covers activities associated with Corrective Action Unit (CAU) 107 of the Federal Facility Agreement and Consent Order (FFACO, 1996 [as amended February 2008]). CAU 107 consists of the following Corrective Action Sites (CASs) located in Areas 1, 2, 3, 4, 5, 9, 10, and 18 of the Nevada Test Site. (1) CAS 01-23-02, Atmospheric Test Site - High Alt; (2) CAS 02-23-02, Contaminated Areas (2); (3) CAS 02-23-03, Contaminated Berm; (4) CAS 02-23-10, Gourd-Amber Contamination Area; (5) CAS 02-23-11, Sappho Contamination Area; (6) CAS 02-23-12, Scuttle Contamination Area; (7) CAS 03-23-24, Seaweed B Contamination Area; (8) CAS 03-23-27, Adze Contamination Area; (9) CAS 03-23-28, Manzanas Contamination Area; (10) CAS 03-23-29, Truchas-Chamisal Contamination Area; (11) CAS 04-23-02, Atmospheric Test Site T4-a; (12) CAS 05-23-06, Atmospheric Test Site; (13) CAS 09-23-06, Mound of Contaminated Soil; (14) CAS 10-23-04, Atmospheric Test Site M-10; and (15) CAS 18-23-02, U-18d Crater (Sulky). Based on historical documentation, personnel interviews, site process knowledge, site visits, photographs, engineering drawings, field screening, analytical results, and the results of data quality objectives process (Section 3.0), closure in place with administrative controls or no further action will be implemented for CAU 107. CAU 107 closure activities will consist of verifying that the current postings required under Title 10 Code of Federal Regulations (CFR) Part 835 are in place and implementing use restrictions (URs) at two sites, CAS 03-23-29 and CAS 18-23-02. The current radiological postings combined with the URs are adequate administrative controls to limit site access and worker dose.

  17. Choosing Actions

    PubMed Central

    Rosenbaum, David A.; Chapman, Kate M.; Coelho, Chase J.; Gong, Lanyun; Studenka, Breanna E.

    2013-01-01

    Actions that are chosen have properties that distinguish them from actions that are not. Of the nearly infinite possible actions that can achieve any given task, many of the unchosen actions are irrelevant, incorrect, or inappropriate. Others are relevant, correct, or appropriate but are disfavored for other reasons. Our research focuses on the question of what distinguishes actions that are chosen from actions that are possible but are not. We review studies that use simple preference methods to identify factors that contribute to action choices, especially for object-manipulation tasks. We can determine which factors are especially important through simple behavioral experiments. PMID:23761769

  18. An Implementation of the Action Space Concept in Behavioral Analysis.

    ERIC Educational Resources Information Center

    Higgs, Gary K.

    The Contact Action Space (CAS) of an individual, or group of individuals, has a significant impact on the location of activities and the organization of the use of space. Beginning with the most basic components of a CAS, the individual behavior pattern element is developed, and operational variations affecting alignment and configuration are…

  19. The Early Development of Object Knowledge: A Study of Infants' Visual Anticipations during Action Observation

    ERIC Educational Resources Information Center

    Hunnius, Sabine; Bekkering, Harold

    2010-01-01

    This study examined the developing object knowledge of infants through their visual anticipation of action targets during action observation. Infants (6, 8, 12, 14, and 16 months) and adults watched short movies of a person using 3 different everyday objects. Participants were presented with objects being brought either to a correct or to an…

  20. Studying Your Own School: An Educator's Guide to Practitioner Action Research, Second Edition

    ERIC Educational Resources Information Center

    Anderson, Gary L.; Herr, Kathryn G.; Nihlen, Ann

    2007-01-01

    Since the publication of the first edition of "Studying Your Own School", practitioner action research has become an established professional development practice in schools and teacher education programs.While the fundamentals of practitioner action research have not changed, the challenges of large scale reform have dramatically altered the…

  1. Student Attitudes toward Affirmative Action in Higher Education: Findings from a National Study.

    ERIC Educational Resources Information Center

    Sax, Linda J.; Arredondo, Marisol

    This report examines the affirmative action attitudes of 204,103 college freshmen from 473 colleges and universities nationwide and from four racial/ethnic groups: Whites, African Americans, Asian Americans, and Mexican Americans. The study addresses how and why student support for affirmative action in higher education admissions depends on how…

  2. Mood as Embodied Action: A Phenomenological Study of Interaction between Self and the Environment

    ERIC Educational Resources Information Center

    Manley, Dolores R.

    2009-01-01

    This phenomenological study explored the interaction between the affective phenomenon of mood (Davidson, et al., 2003) and embodied action (Varela, et al., 1993) experienced during interaction between self and the environment. Exploring the complementarity of mood and embodied action for organizations, teams, or individuals provided insightful…

  3. Iontophoretic study of speed of action of various muscle relaxants.

    PubMed

    Min, J C; Bekavac, I; Glavinovic, M I; Donati, F; Bevan, D R

    1992-08-01

    The speed of action of nondepolarizing muscle relaxants is inversely related to potency. The hypothesis that this effect occurs at the end plate was tested in a frog (Rana pipiens) cutaneous pectoris muscle preparation. Brief acetylcholine pulses (10-100 ms) were applied iontophoretically from a central barrel of a triple-barrelled microelectrode located near an end plate. Long pulses (10-200 s) of muscle relaxant (gallamine, rocuronium, d-tubocurarine, atracurium, vecuronium, pancuronium, and doxacurium) were applied from one of two other barrels. The responses were a voltage change at the end plate, measured with an intracellular electrode. To evaluate potency, intracellular voltage changes following iontophoretic acetylcholine pulses were measured after application of various concentrations of muscle relaxants. The following were the equilibrium dissociation constants, which represent concentration of relaxant for 50% inhibition of response (mean plus or minus standard deviation): gallamine, 4.56 +/- 0.44 microM (n = 5); rocuronium, 0.71 +/- 0.09 microM (n = 6); d-tubocurarine, 0.59 +/- 0.07 microM (n = 4); atracurium, 0.31 +/- 0.03 microM (n = 4); vecuronium, 0.23 +/- 0.02 microM (n = 5); pancuronium, 0.18 +/- 0.03 microM (n = 3); doxacurium, 0.11 +/- 0.03 microM (n = 5). Both onset and offset of effect of muscle relaxant proceeded with an exponential time course.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1353667

  4. Toxicology and carcinogenesis studies of crocidolite asbestos (CAS No. 12001-28-4) in F344/N rats (feed studies). Technical report

    SciTech Connect

    Not Available

    1988-12-01

    Toxicology and carcinogenesis studies of crocidolite asbestos were conducted by administering the chemical to male and female F344/N rats at a concentration of 1% in feed for the lifetime of the animals. Under the conditions of these feed studies, crocidolite asbestos was not overtly toxic and did not cause a carcinogenic response when ingested at a concentration of 1% in the diet by male and female F344/N rats for their lifetime.

  5. 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. PMID:26297561

  6. Knowledge-based discovery for designing CRISPR-CAS systems against invading mobilomes in thermophiles.

    PubMed

    Chellapandi, P; Ranjani, J

    2015-09-01

    Clustered regularly interspaced short palindromic repeats (CRISPRs) are direct features of the prokaryotic genomes involved in resistance to their bacterial viruses and phages. Herein, we have identified CRISPR loci together with CRISPR-associated sequences (CAS) genes to reveal their immunity against genome invaders in the thermophilic archaea and bacteria. Genomic survey of this study implied that genomic distribution of CRISPR-CAS systems was varied from strain to strain, which was determined by the degree of invading mobiloms. Direct repeats found to be equal in some extent in many thermopiles, but their spacers were differed in each strain. Phylogenetic analyses of CAS superfamily revealed that genes cmr, csh, csx11, HD domain, devR were belonged to the subtypes of cas gene family. The members in cas gene family of thermophiles were functionally diverged within closely related genomes and may contribute to develop several defense strategies. Nevertheless, genome dynamics, geological variation and host defense mechanism were contributed to share their molecular functions across the thermophiles. A thermophilic archaean, Thermococcus gammotolerans and thermophilic bacteria, Petrotoga mobilis and Thermotoga lettingae have shown superoperons-like appearance to cluster cas genes, which were typically evolved for their defense pathways. A cmr operon was identified with a specific promoter in a thermophilic archaean, Caldivirga maquilingensis. Overall, we concluded that knowledge-based genomic survey and phylogeny-based functional assignment have suggested for designing a reliable genetic regulatory circuit naturally from CRISPR-CAS systems, acquired defense pathways, to thermophiles in future synthetic biology. PMID:26279704

  7. Structural and dynamic views of the CRISPR-Cas system at the single-molecule level.

    PubMed

    Lee, Seung Hwan; Bae, Sangsu

    2016-04-01

    The CRISPR-Cas system has emerged as a fascinating and important genome editing tool. It is now widely used in biology, biotechnology, and biomedical research in both academic and industrial settings. To improve the specificity and efficiency of Cas nucleases and to extend the applications of these systems for other areas of research, an understanding of their precise working mechanisms is crucial. In this review, we summarize current studies on the molecular structures and dynamic functions of type I and type II Cas nucleases, with a focus on target DNA searching and cleavage processes as revealed by single-molecule observations. [BMB Reports 2016; 49(4): 201-207]. PMID:26923305

  8. Structural and dynamic views of the CRISPR-Cas system at the single-molecule level

    PubMed Central

    Lee, Seung Hwan; Bae, Sangsu

    2016-01-01

    The CRISPR-Cas system has emerged as a fascinating and important genome editing tool. It is now widely used in biology, biotechnology, and biomedical research in both academic and industrial settings. To improve the specificity and efficiency of Cas nucleases and to extend the applications of these systems for other areas of research, an understanding of their precise working mechanisms is crucial. In this review, we summarize current studies on the molecular structures and dynamic functions of type I and type II Cas nucleases, with a focus on target DNA searching and cleavage processes as revealed by single-molecule observations. [BMB Reports 2016; 49(4): 201-207] PMID:26923305

  9. 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.

  10. 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

  11. NTP Toxicology and Carcinogenesis Studies of 3,3'-Dimethoxybenzidine Dihydrochloride (CAS No. 20325-40-0) in F344/N Rats (Drinking Water Studies).

    PubMed

    1990-01-01

    3,3'-Dimethoxybenzidine dihydrochloride is an off-white powder with a melting point of 274 degrees C. 3,3'-Dimethoxybenzidine is used principally as an intermediate in the production of commercial bisazobiphenyl dyes for coloring textiles, paper, plastic, rubber, and leather. In the synthesis of the bisazobiphenyl dyes, the amine groups of 3,3'-dimethoxybenzidine are chemically linked with other aromatic amines. A small quantity of 3,3'-dimethoxybenzidine is also used as an intermediate in the production of o-dianisidine diisocyanate, which is used in isocyanate-based adhesive systems and as a component of polyurethane elastomers. 3,3'-Dimethoxybenzidine dihydrochloride was evaluated in toxicity and carcinogenicity studies as part of the National Toxicology Program's Benzidine Dye Initiative. This Initiative was designed to evaluate the representative benzidine congeners and benzidine congener-derived and benzidine-derived dyes. 3,3'-Dimethoxybenzidine dihydrochloride was nominated for study because of the potential for human exposure during production of bisazobiphenyl dyes and because benzidine, a structurally related chemical, is a known human carcinogen. NTP Toxicology and Carcinogenesis studies were conducted by administering 3,3'-dimethoxybenzidine dihydrochloride (greater than 97.5% pure) in drinking water to groups of F344/N rats of each sex for 14 days, 13 weeks, 9 months, or 21-months. The 21-month studies were intended to last 24 months but were terminated early because of rapidly declining survival due to neoplasia. Studies were performed only in rats because similar studies are being performed in mice at the National Center for Toxicology Research. Genetic toxicology studies were conducted with Salmonella typhimurium, Chinese hamster over (CHO) cells, and Drosophila melanogaster. Fourteen-Day Studies: All rats receiving drinking water concentrations up to 4,500 ppm lived to the end of the studies. Rats that received water containing 4,500 ppm 3

  12. Toxicology and carcinogenesis studies of hexachloroethane (CAS No. 67-72-1) in F344/N rats (gavage studies). Technical report

    SciTech Connect

    Eastin, W.C.

    1989-08-01

    Toxicology and carcinogenesis studies were conducted by administering doses of 0, 10, or 20 mg/kg hexachloroethane in corn oil by gavage 5 days per week for 103 weeks to groups of 50 male rats. Groups of 50 female rats were administered 0, 80, or 160 mg/kg on the same schedule. Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenic activity of hexachloroethane for male F344/N rats, based on the increased incidences of renal neoplasms. The marginally increased incidences of pheochromocytomas of the adrenal gland may have been related to hexachloroethane administration to male rats. There was no evidence of carcinogenic activity of hexachloroethane for female F344/N rats administered 80 or 160 mg/kg by gavage for 103 weeks.

  13. Toxicology and carcinogenesis studies of amosite asbestos (CAS No. 12172-73-5) in F344/N rats (feed studies). Technical report series

    SciTech Connect

    McConnell, E.E.

    1990-11-01

    Carcinogenesis studies of amosite asbestos alone or in combination with the intestinal carcinogen 1,2-dimethylhydrazine dihydrochloride (DMH) were conducted in male and female F344/N rats. Amosite asbestos was administered at a concentration of 1% in pelleted diet for the entire lifetime of the rats, starting with the dams of the study animals. The DMH was administered by gavage at a dose of 7.5 mg/kg for males and 15 mg/kg for females every 14 days, starting at 8 weeks of age, for a total of five doses. The administration of DMH did not affect body weight gain either in amosite-exposed or nonexposed animals. Significant increases in the incidences of C-cell carcinomas of the thyroid gland (untreated control, 11/117; amosite, 50/246, P<0.05; amosite preweaning gavage, 14/100) and of leukemia (38/117; 106/249, P<0.05; 49/100, P<0.01) in male rats were observed in amosite-exposed groups. However, the biologic significance of the C-cell carcinomas in relation to amosite asbestos exposure is discounted because of a lack of significance when C-cell adenomas and carcinomas were combined and because the positive effect was not observed in the amosite preweaning gavage group. DMH caused a high incidence (62%-74%) of intestinal neoplasia in amosite-exposed and nonexposed groups. Neither an enchanced carcinogenic nor a protective effect was demonstrated by exposure to amosite asbestos. Under the conditions of these feed studies, amosite asbestos was not overtly toxic, did not affect survival, and was not carcinogenic when ingested at a concentration of 1% in the diet by male or female F344/N rats. The cocarcinogenic studies using DMH were considered inadequate because of the high incidence of DMH-induced intestinal neoplasia in both the amosite asbestos-exposed and nonexposed groups.

  14. Lifetime carcinogenesis studies of chrysotile asbestos (CAS No. 12001-29-5) in syrian golden hamsters (feed studies). Technical report series

    SciTech Connect

    Not Available

    1990-07-01

    Carcinogenesis studies of short range (SR), intermediate range (IR) or intermediate range chrysotile asbestos in combination with the intestinal carcinogen 1,2-dimethylhydrazine dihydrochloride (DMH) were conducted with male and female Syrian golden hamsters. Both forms of chrysotile asbestos were administered at a concentration of 1% in pelleted diet for the entire lifetime of the hamsters, starting with mothers of the test animals. Group sizes varied from 125 to 253. Starting at 6 weeks of age, male and female hamsters in the intermediate range chrysotile/DMH study were given oral doses of DMH (4 mg/kg) every other week for a total of 5 doses. There was no adverse effect on body weight gain or survival by either form of asbestos or by asbestos in combination with DMH. Under the conditions of these studies, neither short range chrysotile nor intermediate range chrysotile asbestos was carcinogenic when ingested at 1% levels in the diet by male and female Syrian golden hamsters. While there were increases in the rates of adrenal cortical adenomas in male and female hamsters exposed to intermediate range chrysotile asbestos compared with pooled control groups, these incidence rates were not different when compared with the concurrent control groups. Additionally, the biologic importance of adrenal tumors in the absence of target organ (gastrointestinal tract) neoplasia is questionable.

  15. Toxicology and carcinogenesis studies of indium phosphide (CAS No. 22398-90-7) in F344/N rats and B6C3F1 mice (inhalation studies).

    PubMed

    2001-07-01

    Indium phosphide is used to make semiconductors,injection lasers, solar cells, photodiodes, and light-emittingdiodes. Indium phosphide was nominated for study because of its widespread use in the microelectronics industry, the potential for worker exposure,and the absence of chronic toxicity data. Male and female F344/N rats and B6C3F1 mice were exposed to indium phosphide (greater than 99% pure) by inhalation for 14 weeks or 2 years. The frequency of micronuclei was determined in the peripheral blood of mice exposed to indium phosphide for 14 weeks. 14-WEEK STUDY IN RATS: Groups of 10 male and 10 female rats were exposed to particulate aerosols of indium phosphide with amass median aerodynamic diameter of approximately 1.2 microm at concentrations of 0, 1, 3, 10, 30, or 100 mg/m3 by inhalation, 6 hours per day, 5 days per week (weeks 1 through 4 and weeks 10 through 14) or 7 days per week (weeks 5 through 9) to accommodate a concurrent teratology study. One male in the 100 mg/m3 group died before the end of the study. Body weight gains of all males and females exposed to 100 mg/m3 were less than those of the chamber controls. As a result of indium phosphide exposure, the lungs of all exposed rats had a gray to black discoloration and were significantly enlarged, weighing 2.7- to 4.4-fold more than those of the chamber controls. Indium phosphide particles were observed throughout the respiratory tract and in the lung-associated lymph nodes. A spectrum of inflammatory and proliferative lesions generally occurred in the lungs of all exposed groups of rats and consisted of alveolar proteinosis, chronic inflammation, interstitial fibrosis, and alveolar epithelial hyperplasia. Pulmonary inflammation was attended by increased leukocyte and neutrophil counts in the blood. The alveolar proteinosis was the principal apparent reason for the increase in lung weights. Indium phosphide caused inflammation at the base of the epiglottis of the larynx and hyperplasia of the

  16. Teaching in Action: Case Studies from Second Language Classrooms.

    ERIC Educational Resources Information Center

    Richards, Jack C., Ed.

    The 76 case studies presented here describe how teachers of English as a second language (ESL) respond to problems they encounter in teaching. Each case study describes the context in which the teacher is working, the problem, and the teacher's response. Following each case study is a brief commentary by a teacher educator who reflects that type…

  17. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 7 2014-10-01 2014-10-01 false Types of CAS coverage... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  18. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Types of CAS coverage... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  19. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 7 2013-10-01 2012-10-01 true Types of CAS coverage. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  20. Assisting Students' Cognitive Strategies with the Use of CAS

    ERIC Educational Resources Information Center

    Sarvari, Csaba; Lavicza, Zsolt; Klincsik, Mihaly

    2010-01-01

    This paper examines various cognitive strategies applied while CAS (Computer Algebra System) are used in undergraduate-level engineering mathematics teaching and learning. We posed some questions in relation to such CAS use: What kind of tools can CAS offer to enhance different cognitive strategies of students? How can the use of CAS widen the…

  1. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 7 2012-10-01 2012-10-01 false Types of CAS coverage... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  2. NTP Toxicology and Carcinogenesis Studies of Nitromethane (CAS No. 75-52-5) in F344/N Rats and B6C3F1 Mice (Inhalation Studies).

    PubMed

    1997-02-01

    Nitromethane is used as a rocket and engine fuel; as a synthesis intermediate for agricultural fumigants, biocides, and other products; as a solvent; and as an explosive in mining, oil-well drilling, and seismic exploration. It has been detected in air, in surface and drinking water, and in cigarette smoke. Nitromethane was studied because of the potential for widespread human exposure and because it is structurally related to the carcinogens 2-nitropropane and tetranitromethane. Male and female F344/N rats and B6C3F1 mice received nitromethane (purity 98% or greater) by inhalation for 16 days, 13 weeks, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, and peripheral blood erythrocytes of mice. 16-DAY STUDY IN RATS: Groups of five male and five female rats were exposed to 0, 94, 188, 375, 750, or 1,500 ppm nitromethane by inhalation, 6 hours per day, 5 days per week, for 16 days. All rats survived until the end of the study. The mean body weight gain of male rats in the 1,500 ppm group was slightly but significantly less than that of the controls; the final mean body weights and mean body weight gains of exposed females were similar to those of the controls. Clinical findings in all male and female rats in the 1,500 ppm groups included increased preening, rapid breathing, hyperactivity early in the study, and hypoactivity and loss of coordination in the hindlimbs near the end of the study. The relative liver weights of all exposed groups of male rats and the absolute and relative liver weights of females exposed to 375 ppm or greater were significantly greater than those of the controls. Minimal to mild degeneration of the olfactory epithelium was observed in the nose of males and females exposed to 375 ppm or greater. Sciatic nerve degeneration was present in all male and female rats exposed to 375 ppm or greater; rats exposed to 750 or 1,500 ppm also had reduced myelin around sciatic axons. 16

  3. Toxicology and carcinogenesis studies of tetralin (CAS No. 119-64-2) in F344/N rats and B6C3F1 mice (inhalation studies).

    PubMed

    2011-04-01

    Tetralin is used as an industrial solvent primarily for naphthalene, fats, resins, oils, and waxes; as a solvent and stabilizer for shoe polishes and floor waxes; as a solvent for pesticides, rubber, asphalt, and aromatic hydrocarbons (e.g., anthracene); as a dye solvent carrier in the textile industry; as a substitute for turpentine in lacquers, paints, and varnishes; in paint thinners and as a paint remover; in alkali-resistant lacquers for cleaning printing ink from rollers and type; as a constituent of motor fuels and lubricants; for the removal of naphthalene in gas distribution systems; and as an insecticide for clothes moths. Tetralin was nominated by the National Cancer Institute for carcinogenicity and disposition studies because of its structure, high production volume, and high potential for worker and consumer exposure. Male and female F344/N rats and B6C3F1 mice were exposed to tetralin (at least 97% pure) by inhalation for 2 weeks, 3 months, or 2 years; male NCI Black Reiter (NBR) rats were exposed to tetralin by inhalation for 2 weeks. Male NBR rats do not produce 2u-globulin; the NBR rats were included to study the relationship of 2u-globulin and renal lesion induction. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, and mouse peripheral blood erythrocytes. 2-WEEK STUDY IN RATS: Groups of five male (F344/N and NBR) and five female (F344/N) rats were exposed to tetralin at air concentrations of 0, 7.5, 15, 30, 60, or 120 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 12 exposures. All rats survived to the end of the studies. The final mean body weight of female rats exposed to 120 ppm and mean body weight gains of female rats exposed to 30 ppm or greater were significantly less than those of the chamber controls. Final mean body weights of exposed groups of male NBR rats and mean body weight gains of all exposed groups of male rats were significantly less than those of the chamber controls. Dark

  4. NTP Technical Report on the comparative toxicity studies of allyl acetate (CAS No. 591-87-7), allyl alcohol (CAS No. 107-18-6) and acrolein (CAS No. 107-02-8) administered by gavage to F344/N rats and B6C3F1 mice.

    PubMed

    Irwin, Rick D

    2006-07-01

    Allyl acetate, allyl alcohol, and acrolein are used in the manufacture of detergents, plastics, pharmaceuticals, and chemicals and as agricultural agents and food additives. Male and female F344/N rats and B6C3F(1) mice received allyl acetate, allyl alcohol, or acrolein by gavage for 14 weeks. Genetic toxicology studies were conducted in Salmonella typhimurium, Drosophila melanogaster, cultured Chinese hamster ovary cells, rat bone marrow erythrocytes, and mouse peripheral blood erythrocytes. Groups of 10 male and 10 female rats were administered 0, 6, 12, 25, 50, or 100 mg allyl acetate/kg body weight, 0, 1.5, 3, 6, 12, or 25 mg/kg allyl alcohol, or 0, 0.75, 1.25, 2.5, 5, or 10 mg/kg acrolein in 0.5% methylcellulose by gavage, 5 days per week for 14 weeks. Groups of 10 male and 10 female mice were administered 0, 8, 16, 32, 62.5, or 125 mg/kg allyl acetate, 0, 3, 6, 12, 25, or 50 mg/kg allyl alcohol, or 0, 1.25, 2.5, 5, 10, or 20 mg/kg acrolein in 0.5% methylcellulose by gavage, 5 days per week for 14 weeks. In the allyl acetate rat study, all males and females in the 100 mg/kg groups died or were killed moribund by day 8; there were no other deaths. In the allyl alcohol study, all rats survived to the end of the study except one 6 mg/kg female. In the acrolein rat study, eight males and eight females in the 10 mg/kg groups died by week 9 of the study. Two males in the 2.5 and 5 mg/kg groups and one or two females in the 1.25, 2.5, and 5 mg/kg groups also died early; two of these deaths were gavage accidents. In the allyl acetate mouse study, all males and females in the 125 mg/kg group died during the first week of the study. All other early deaths, except five 62.5 mg/kg males and one 32 mg/kg female, were gavage accidents. In the allyl alcohol mouse study, one 50 mg/kg female died due to a gavage accident; all other animals survived to the end of the study. In the acrolein mouse study, all males and females administered 20 mg/kg died during the first week of

  5. Toxicology and carcinogenesis studies of 1-bromopropane (CAS No. 106-94-5) in F344/N rats and B6C3F1 mice (inhalation studies).

    PubMed

    2011-08-01

    In the early to mid 1990s, 1-bromopropane was used primarily as an intermediate in the production of pesticides, quaternary ammonium compounds, flavors and fragrances, pharmaceuticals, and other chemicals in well-controlled, closed processes. In the mid to late 1990s, it was introduced as a less toxic replacement for methylene chloride in emissive applications such as vapor and immersion degreasing operations and critical cleaning of electronics and metals. 1-Bromopropane was also introduced as a nonflammable, nontoxic, fast-drying, and inexpensive solvent for adhesive resins, and has been marketed as a replacement for ozone depleting refrigerants. 1-Bromopropane was nominated for study by the Occupational Safety and Health Administration based on the potential for widespread occupational and environmental exposure and a lack of toxicity and carcinogenicity data. Male and female F344/N rats and B6C3F1 mice were exposed to 1-bromopropane (99% or greater pure) by inhalation for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and Escherichia coli and mouse peripheral blood. 2-WEEK STUDY IN RATS: Groups of five male and five female rats were exposed to 1-bromopropane vapor at concentrations of 0, 125, 250, 500, 1,000, or 2,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 16 days. All rats survived to the end of the study except one 500 ppm male. Mean body weights of 2,000 ppm rats were significantly less than those of the chamber controls. The absolute kidney weight of 1,000 ppm males, relative kidney weights of all exposed groups of males, and absolute and relative kidney weights of all exposed groups of females were significantly increased. The absolute and relative liver weights of 1,000 ppm males, relative liver weights of 500 and 2,000 ppm males, and absolute and relative liver weights of 500 ppm or greater females were significantly increased. Nasal lesions included suppurative inflammation in

  6. NTP Toxicology and Carcinogenesis Studies 2-Butoxyethanol (CAS NO. 111-76-2) in F344/N Rats and B6C3F1 Mice (Inhalation Studies).

    PubMed

    2000-03-01

    2-Butoxyethanol is a member of a family of ethylene glycol monoalkyl ethers. It is used extensively as a solvent in surface coatings such as lacquers, enamels, varnishes, and latex paint; in paint thinners, paint stripping formulations, and inks; and in degreasers and industrial and household cleaners. 2-Butoxyethanol was nominated for study because of its widespread use in industrial and consumer applications, the potential for exposure to workers and the general population, and the absence of chronic toxicity data. Male and female F344/N rats and B6C3F1 mice were exposed to 2-butoxyethanol (greater than 99% pure) by inhalation (primary route of human exposure) for 14 weeks or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, and the bone marrow of male F344/N rats and B6C3F1 mice. 14-WEEK STUDY IN RATS: Groups of 10 male and 10 female rats were exposed to 2-butoxyethanol by inhalation at concentrations of 0, 31, 62.5, 125, 250, or 500 ppm, 6 hours per day, 5 days per week for 14 weeks. One female rat in the 250 ppm group was killed moribund during week 8; four females in the 500 ppm group were killed moribund during week 1 and one during week 5. Final mean body weights of females exposed to 500 ppm were significantly less than those of the chamber controls. Clinical findings included abnormal breathing, pallor, red urine stains, nasal and eye discharge, lethargy, and increased salivation and/or lacrimation. Due to vascular thrombosis and infarction in the tail vertebrae of 500 ppm female rats, the tails became necrotic and either sloughed off or were chewed off. The primary effect on the hematopoietic system was an anemia characterized as macrocytic, normochromic, and regenerative in males exposed to 125 ppm or greater and, to a greater extent, in all exposed groups of females. Compared to the chamber controls, kidney weights of males exposed to 500 ppm and females exposed to 125 ppm or greater and

  7. Toxicology and carcinogenesis studies of pulegone (CAS No. 89-82-7) in F344/N rats and B6C3F1 mice (gavage studies).

    PubMed

    2011-08-01

    Several essential oils contain pulegone and are used for flavoring foods, drinks, and dental products, as fragrance agents, and in herbal medicines. Pulegone was nominated for study by the National Institute of Environmental Health Sciences based on the potential for human exposure and the absence of carcinogenicity data. Male and female F344/N rats and B6C3F1 mice received pulegone (approximately 96% pure) by gavage for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, and mouse peripheral blood erythrocytes. 2-WEEK STUDY IN RATS: Groups of five male and five female rats were administered 0, 37.5, 75, 150, 300, or 600 mg pulegone/kg body weight in corn oil by gavage, 5 days per week for 16 days. All male rats and nearly all female rats in the 300 and 600 mg/kg groups died prior to the end of the study. All moribund sacrifices and early deaths were attributed to liver toxicity. Mean body weight gains of males administered 37.5 or 150 mg/kg were significantly less than that of the vehicle controls. Clinical findings in 300 and 600 mg/kg rats included nasal/eye discharge, thinness, lethargy, and ruffled fur. Liver and kidney weights of dosed groups of females were generally significantly greater than those of the vehicle control group. The incidences of necrosis and cytoplasmic vacuolization of the liver in 300 and 600 mg/kg males and females were significantly greater than those in the vehicle control groups. 2-WEEK STUDY IN MICE: Groups of five male and five female mice were administered 0, 18.75, 37.5, 75, 150, or 300 mg pulegone/kg body weight in corn oil by gavage, 5 days per week for 16 days. Four females and one male in the 300 mg/kg groups died by study day 5. All early deaths were attributed to liver toxicity. Mean body weights of the dosed groups were similar to those of the vehicle controls. Clinical findings were observed only in 300 mg/kg mice and included thinness, lethargy, and ruffled

  8. NTP Toxicology and Carcinogenesis Studies of 1,3-Butadiene (CAS No. 106-99-0) in B6C3F1 Mice (Inhalation Studies).

    PubMed

    1984-08-01

    1,3-Butadiene is used as an intermediate in the production of elastomers, polymers, and other chemicals. Of the 1,3-butadiene used in 1978, 44% was used to manufacture styrene-butadiene rubber (a substitute for natural rubber, produced by copolymerization of 1,3-butadiene with styrene), and 19% was used to produce polybutane elastomer (a substance that increases resistance of tire products to wear, heat degradation, and blowouts). Chloroprene monomer, derived from 1,3-butadiene, is used exclusively to manufacture neoprene elastomers for non-tire and latex applications. Commercial nitrile rubber, used largely in rubber hoses, seals, and gaskets for automobiles, is a copolymer of 1,3-butadiene and acrylonitrile. Acrylonitrile- butadiene- styrene resins, usually containing 20%-30% 1,3-butadiene by weight, are used to make parts for automobiles and appliances. Other polymer uses include specialty polybutadiene polymers, thermoplastic elastomers, nitrile barrier resins, and K resins(R). 1,3-Butadiene is used as an intermediate in the production of a variety of industrial chemicals, including two fungicides, captan and captofol. It is approved by the U.S. Food and Drug Administration for use in the production of adhesives used in articles for packaging, transporting, or holding food; in components of paper and paperboard that are in contact with dry food; and as a modifier in the production of semigrid and rigid vinyl chloride plastic food-contact articles. No information was located on the levels of monomer or on its elution rate from any of the commercially available polymers. It is not known if unreacted 1,3-butadiene migrated from packaging materials. Male and female B6C3F1 mice were exposed to air containing 1,3-butadiene (greater than 99% pure) at concentrations of 0-8,000 ppm in 15-day and 14-week inhalation studies. In the 15-day studies, survival was unaffected by dose, and no pathologic effects were observed; slight decreases in mean body weight occurred at the

  9. NTP Toxicology and Carcinogenesis Studies of Coumarin (CAS No. 91-64-5) in F344/N Rats and B6C3F1 Mice (Gavage Studies).

    PubMed

    1993-09-01

    Coumarin is the basic structure of numerous naturally occurring compounds with important and diverse physiological activities. More than a thousand coumarin derivatives have been described, varying from simple coumarins containing alkyl and hydroxyl side chains to complex coumarins with benzoyl, furanoyl, pyranoyl, or alkylphosphorothionyl substituents. Coumarin and 3,4-dihydrocoumarin were nominated by the Food and Drug Administration and the National Cancer Institute for study because of the widespread use of coumarin in perfumes, cosmetics, and other products as a fragrance, continued interest in coumarin compounds as flavor-enhancing agents for foods, and the interest in structure-activity relationships of this important group of compounds. Coumarin is believed to be metabolized to a 3,4-epoxide intermediate, which may be responsible for its toxic effects, while 3,4-dihydrocoumarin, which lacks the 3,4-double bond, is not considered likely to form an epoxide intermediate. Toxicity and carcinogenicity studies were conducted by administering coumarin (97% pure) in corn oil by gavage to groups of male and female F344/N rats and B6C3F1 mice for 16 days, 13 weeks, and 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, Drosophila melanogaster, and B6C3F1 mice. 16-DAY STUDY IN RATS: Groups of five male and five female rats received coumarin in corn oil by gavage at doses of 0, 25, 50, 100, 200, or 400 mg per kg body weight, 5 days a week for a total of 12 doses in a 16-day period. All female rats and four male rats receiving 400 mg/kg died. The mean body weight gains and final mean body weights of surviving dosed male and female rats were similar to those of the controls. There were no clinical signs of organ-specific toxicity, and there was no evidence of impaired blood coagulation from measurements of capillary clotting time or prothrombin and activated partial thromboplastin time. 16-DAY STUDY IN MICE

  10. Mathematics in Action: Two New Zealand Case Studies.

    ERIC Educational Resources Information Center

    Ryan, David M.

    Mathematics is playing an increasingly important role in business and industry. In this paper we present two case studies to illustrate the power and impact of mathematics in two important practical applications in New Zealand. The first case study describes the development of a mathematical optimization model to maximize the value of aluminum…

  11. The Girl Child and the Family: An Action Research Study.

    ERIC Educational Resources Information Center

    Anandalakshmy, S., Comp.

    This report describes a nationwide study of female children and the family in rural India. The objectives of the study were to generate data on the situation of female children; to identify the major problems related to their status; to start a series of programs to help remedy those shortcomings; and to assist communities, and women in…

  12. Educational Cost Analysis in Action: Case Studies for Planners -- II.

    ERIC Educational Resources Information Center

    Coombs, Philip H.; Hallak, Jacques

    This document is the second in a series of three documents, which together contain 27 case studies on the uses of cost analysis in educational planning. The case studies are presented to help planners and administrators see how cost analysis can be used to improve the efficiency of their educational systems, or to get the best value existing…

  13. An fMRI study of perception and action in deaf signers.

    PubMed

    Okada, Kayoko; Rogalsky, Corianne; O'Grady, Lucinda; Hanaumi, Leila; Bellugi, Ursula; Corina, David; Hickok, Gregory

    2016-02-01

    Since the discovery of mirror neurons, there has been a great deal of interest in understanding the relationship between perception and action, and the role of the human mirror system in language comprehension and production. Two questions have dominated research. One concerns the role of Broca's area in speech perception. The other concerns the role of the motor system more broadly in understanding action-related language. The current study investigates both of these questions in a way that bridges research on language with research on manual actions. We studied the neural basis of observing and executing American Sign Language (ASL) object and action signs. In an fMRI experiment, deaf signers produced signs depicting actions and objects as well as observed/comprehended signs of actions and objects. Different patterns of activation were found for observation and execution although with overlap in Broca's area, providing prima facie support for the claim that the motor system participates in language perception. In contrast, we found no evidence that action related signs differentially involved the motor system compared to object related signs. These findings are discussed in the context of lesion studies of sign language execution and observation. In this broader context, we conclude that the activation in Broca's area during ASL observation is not causally related to sign language understanding. PMID:26796716

  14. 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...

  15. Closure Report for Corrective Action Unit 540: Spill Sites, Nevada Test Site, Nevada, Rev. No.: 0

    SciTech Connect

    McClure, Lloyd

    2006-10-01

    This Closure Report (CR) presents information supporting the closure of Corrective Action Unit (CAU) 540: Spill Sites, Nevada Test Site, Nevada. This CR complies with the requirements of the 'Federal Facility Agreement and Consent Order' (1996) that was agreed to by the State of Nevada, the U.S. Department of Energy, and the U.S. Department of Defense. Corrective Action Unit 540 is located within Areas 12 and 19 of the Nevada Test Site and is comprised of the following Corrective Action Sites (CASs): CAS 12-44-01, ER 12-1 Well Site Release; CAS 12-99-01, Oil Stained Dirt; CAS 19-25-02, Oil Spill; CAS 19-25-04, Oil Spill; CAS 19-25-05, Oil Spill; CAS 19-25-06, Oil Spill; CAS 19-25-07, Oil Spill; CAS 19-25-08, Oil Spills (3); and CAS 19-44-03, U-19bf Drill Site Release. The purpose of this CR is to provide documentation supporting recommendations of no further action for the CASs within CAU 540. To achieve this, the following actions were performed: (1) Reviewed the current site conditions, including the concentration and extent of contamination; (2) Performed closure activities to address the presence of substances regulated by 'Nevada Administrative Code' 445A.2272 (NAC, 2002); and (3) Documented Notice of Completion and closure of CAU 540 issued by the Nevada Division of Environmental Protection.

  16. A Multidisciplinary Osteoporosis Service-Based Action Research Study

    ERIC Educational Resources Information Center

    Whitehead, Dean; Keast, John; Montgomery, Val; Hayman, Sue

    2004-01-01

    Objective: To investigate an existing Trust-based osteoporosis service's preventative activity, determine any issues and problems and use this data to reorganise the service, as part of a National Health Service Executive/Regional Office-commissioned and funded study. Setting: A UK Hospital Trust's Osteoporosis Service. Design & Method: A…

  17. From Numbers to Action: A Preliminary Study of Retention.

    ERIC Educational Resources Information Center

    Greenlee, Shelia Parker; Greenlee, Harry

    This study examined retention at Christopher Newport University (CNU) in Newport News, Virginia, focusing on the demographic characteristics of those students who left the university as well as the reasons why they left. A total of 159 students who had attended CNU during the 1994-95 academic year but who did not re-enroll in the fall of 1995 were…

  18. Reading Comprehension across Different Genres: An Action Research Study

    ERIC Educational Resources Information Center

    Lubawski, Michael; Sheehan, Caitlyn

    2010-01-01

    Reading comprehension is often a forgotten skill in secondary classrooms. Teachers of older students assign readings of different styles and levels, but don't teach the skills necessary to process different kinds of texts. This study examined which strategies are effective in giving students the skills necessary to read for understanding by…

  19. Distributed Curriculum Leadership in Action: A Hong Kong Case Study

    ERIC Educational Resources Information Center

    Law, Edmond; Galton, Maurice; Wan, Sally

    2010-01-01

    This study was designed primarily to investigate the impact of school-based curriculum development teams on teacher development within the tradition of school-based curriculum development. The results are expected to provide valuable insights for teachers, school management and policy making. Teacher interviews in a primary school in Hong Kong…

  20. Rural Action: A Collection of Community Work Case Studies.

    ERIC Educational Resources Information Center

    Henderson, Paul, Ed.; Francis, David, Ed.

    This book contains 10 case studies of rural community development in England, Wales, Scotland, Ireland, and Catalonia, as seen from the perspective of community-work practitioners. Development projects encompassed such activities as promotion of tourism, establishment of community centers, vocational training for school dropouts, adult community…

  1. Corrective Action Investigation Plan for Corrective Action Unit 137: Waste Disposal Sites, Nevada Test Site, Nevada, Rev. No.:0

    SciTech Connect

    Wickline, Alfred

    2005-12-01

    This Corrective Action Investigation Plan (CAIP) contains project-specific information including facility descriptions, environmental sample collection objectives, and criteria for conducting site investigation activities at Corrective Action Unit (CAU) 137: Waste Disposal Sites. This CAIP has been developed in accordance with the ''Federal Facility Agreement and Consent Order'' (FFACO) (1996) that was agreed to by the State of Nevada, the U.S. Department of Energy (DOE), and the U.S. Department of Defense. Corrective Action Unit 137 contains sites that are located in Areas 1, 3, 7, 9, and 12 of the Nevada Test Site (NTS), which is approximately 65 miles (mi) northwest of Las Vegas, Nevada (Figure 1-1). Corrective Action Unit 137 is comprised of the eight corrective action sites (CASs) shown on Figure 1-1 and listed below: (1) CAS 01-08-01, Waste Disposal Site; (2) CAS 03-23-01, Waste Disposal Site; (3) CAS 03-23-07, Radioactive Waste Disposal Site; (4) CAS 03-99-15, Waste Disposal Site; (5) CAS 07-23-02, Radioactive Waste Disposal Site; (6) CAS 09-23-07, Radioactive Waste Disposal Site; (7) CAS 12-08-01, Waste Disposal Site; and (8) CAS 12-23-07, Waste Disposal Site. The Corrective Action Investigation (CAI) will include field inspections, radiological surveys, geophysical surveys, sampling of environmental media, analysis of samples, and assessment of investigation results, where appropriate. Data will be obtained to support corrective action alternative evaluations and waste management decisions. The CASs in CAU 137 are being investigated because hazardous and/or radioactive constituents may be present in concentrations that could potentially pose a threat to human health and the environment. Existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives for the CASs. Additional information will be generated by conducting a CAI before evaluating and selecting corrective action

  2. V723 Cas a borderline classical nova

    NASA Astrophysics Data System (ADS)

    Friedjung, M.; Iijima, T.

    2002-11-01

    V723 Cas had a light curve similar to that of HR Del before maximum, with a very slow pre-maximum rise, explained according to [2] by the presence of an optically thin wind before maximum unlike the optically thick wind generally seen for classical novae after maximum. Examination of the Fe II emission lines by the SAC method, is compatible with this also having been the case for V723 Cas.

  3. Rhetoric to action: a study of stakeholder perceptions of aging well in two local communities.

    PubMed

    Everingham, Jo-Anne; Lui, Chi-Wai; Bartlett, Helen; Warburton, Jeni; Cuthill, Michael

    2010-11-01

    This qualitative study of local perceptions of policy goals and action in relation to aging reports 31 stakeholder interviews within 2 Australian communities exploring (a) the meaning of aging well; and (b) preferred policy actions to achieve positive aging outcomes. Findings suggest that community perceptions of aging well are broadly consistent with the goals of national and international policy frameworks in focusing on 3 dimensions--health, social engagement, and security. Further, participants believe that achievement of positive aging outcomes requires a mix of self-help, community action, and government intervention--particularly government support and encouragement for aging well initiatives. PMID:20972930

  4. Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems.

    PubMed

    Leenay, Ryan T; Maksimchuk, Kenneth R; Slotkowski, Rebecca A; Agrawal, Roma N; Gomaa, Ahmed A; Briner, Alexandra E; Barrangou, Rodolphe; Beisel, Chase L

    2016-04-01

    CRISPR-Cas adaptive immune systems in prokaryotes boast a diversity of protein families and mechanisms of action, where most systems rely on protospacer-adjacent motifs (PAMs) for DNA target recognition. Here, we developed an in vivo, positive, and tunable screen termed PAM-SCANR (PAM screen achieved by NOT-gate repression) to elucidate functional PAMs as well as an interactive visualization scheme termed the PAM wheel to convey individual PAM sequences and their activities. PAM-SCANR and the PAM wheel identified known functional PAMs while revealing complex sequence-activity landscapes for the Bacillus halodurans I-C (Cascade), Escherichia coli I-E (Cascade), Streptococcus thermophilus II-A CRISPR1 (Cas9), and Francisella novicida V-A (Cpf1) systems. The PAM wheel was also readily applicable to existing high-throughput screens and garnered insights into SpyCas9 and SauCas9 PAM diversity. These tools offer powerful means of elucidating and visualizing functional PAMs toward accelerating our ability to understand and exploit the multitude of CRISPR-Cas systems in nature. PMID:27041224

  5. Photometry of stars in the Cas OB5 Associations

    NASA Astrophysics Data System (ADS)

    Tanriver, Mehmet; Keskin, Ahmet

    2016-07-01

    OB associations are a grouping of very young associations, contain 10-100 very hot massive stars, spectral types O and B. Also, the OB associations contain low and intermediate mass stars, too. Association members are believed to form within the same small volume inside a giant molecular cloud. Once the surrounding dust and gas is blown away, the remaining stars become not tied up and begin to drift separately. It is believed that the majority of all stars in the Milky Way were formed in OB associations. O type stars are short-lived, and will be at an end as supernovae after roundly a million years. OB associations are generally only a few million years in age or less. In this study, the photometry of UU Cas and field star which been Cas OB5 association member was carried out. Light curves and color diagrams are given in the study.

  6. Characterization of Cas9-Guide RNA Orthologs.

    PubMed

    Braff, Jonathan L; Yaung, Stephanie J; Esvelt, Kevin M; Church, George M

    2016-01-01

    In light of the multitude of new Cas9-mediated functionalities, the ability to carry out multiple Cas9-enabled processes simultaneously and in a single cell is becoming increasingly valuable. Accomplishing this aim requires a set of Cas9-guide RNA (gRNA) pairings that are functionally independent and insulated from one another. For instance, two such protein-gRNA complexes would allow for concurrent activation and editing at independent target sites in the same cell. The problem of establishing orthogonal CRISPR systems can be decomposed into three stages. First, putatively orthogonal systems must be identified with an emphasis on minimizing sequence similarity of the Cas9 protein and its associated RNAs. Second, the systems must be characterized well enough to effectively express and target the systems using gRNAs. Third, the systems should be established as orthogonal to one another by testing for activity and cross talk. Here, we describe the value of these orthogonal CRISPR systems, outline steps for selecting and characterizing potentially orthogonal Cas9-gRNA pairs, and discuss considerations for the desired specificity in Cas9-coupled functions. PMID:27140923

  7. The cerebellum in action: a simulation and robotics study.

    PubMed

    Hofstötter, Constanze; Mintz, Matti; Verschure, Paul F M J

    2002-10-01

    The control or prediction of the precise timing of events are central aspects of the many tasks assigned to the cerebellum. Despite much detailed knowledge of its physiology and anatomy, it remains unclear how the cerebellar circuitry can achieve such an adaptive timing function. We present a computational model pursuing this question for one extensively studied type of cerebellar-mediated learning: the classical conditioning of discrete motor responses. This model combines multiple current assumptions on the function of the cerebellar circuitry and was used to investigate whether plasticity in the cerebellar cortex alone can mediate adaptive conditioned response timing. In particular, we studied the effect of changes in the strength of the synapses formed between parallel fibres and Purkinje cells under the control of a negative feedback loop formed between inferior olive, cerebellar cortex and cerebellar deep nuclei. The learning performance of the model was evaluated at the circuit level in simulated conditioning experiments as well as at the behavioural level using a mobile robot. We demonstrate that the model supports adaptively timed responses under real-world conditions. Thus, in contrast to many other models that have focused on cerebellar-mediated conditioning, we investigated whether and how the suggested underlying mechanisms could give rise to behavioural phenomena. PMID:12405996

  8. Activity-Based Teaching in Social Studies Education: An Action Research

    ERIC Educational Resources Information Center

    Akkus, Zekerya

    2015-01-01

    The aim of this study was to determine pre-service social studies teachers' skills to plan and apply the activity-based teaching and contribute to their development of these skills. In the study, the action research design of qualitative research was used. The sample of the study consisted of 6 pre-service teachers who were 4th year students at…

  9. CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape

    PubMed Central

    Wang, Gang; Zhao, Na; Berkhout, Ben; Das, Atze T

    2016-01-01

    Several recent studies demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1 replication by harnessing T cells with Cas9 and antiviral gRNAs. However, the virus rapidly and consistently escaped from this inhibition. Sequencing of the HIV-1 escape variants revealed nucleotide insertions, deletions, and substitutions around the Cas9/gRNA cleavage site that are typical for DNA repair by the nonhomologous end-joining pathway. We thus demonstrate the potency of CRISPR-Cas9 as an antiviral approach, but any therapeutic strategy should consider the viral escape implications. PMID:26796669

  10. 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

  11. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation.

    PubMed

    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

  12. Microcalorimetry studies of the antimicrobial actions of Aconitum alkaloids*

    PubMed Central

    Shi, Yan-bin; Liu, Lian; Shao, Wei; Wei, Ting; Lin, Gui-mei

    2015-01-01

    The metabolic activity of organisms can be measured by recording the heat output using microcalorimetry. In this paper, the total alkaloids in the traditional Chinese medicine Radix Aconiti Lateralis were extracted and applied to Escherichia coli and Staphylococcus aureus. The effect of alkaloids on bacteria growth was studied by microcalorimetry. The power-time curves were plotted with a thermal activity monitor (TAM) air isothermal microcalorimeter and parameters such as growth rate constant (μ), peak-time (Tm), inhibitory ratio (I), and enhancement ratio (E) were calculated. The relationships between the concentration of Aconitum alkaloids and μ of E. coli or S. aureus were discussed. The results showed that Aconitum alkaloids had little effect on E. coli and had a potentially inhibitory effect on the growth of S. aureus. PMID:26238544

  13. Microcalorimetry studies of the antimicrobial actions of Aconitum alkaloids.

    PubMed

    Shi, Yan-bin; Liu, Lian; Shao, Wei; Wei, Ting; Lin, Gui-mei

    2015-08-01

    The metabolic activity of organisms can be measured by recording the heat output using microcalorimetry. In this paper, the total alkaloids in the traditional Chinese medicine Radix Aconiti Lateralis were extracted and applied to Escherichia coli and Staphylococcus aureus. The effect of alkaloids on bacteria growth was studied by microcalorimetry. The power-time curves were plotted with a thermal activity monitor (TAM) air isothermal microcalorimeter and parameters such as growth rate constant (μ), peak-time (Tm), inhibitory ratio (I), and enhancement ratio (E) were calculated. The relationships between the concentration of Aconitum alkaloids and μ of E. coli or S. aureus were discussed. The results showed that Aconitum alkaloids had little effect on E. coli and had a potentially inhibitory effect on the growth of S. aureus. PMID:26238544

  14. Canceling planned action: an FMRI study of countermanding saccades.

    PubMed

    Curtis, Clayton E; Cole, Michael W; Rao, Vikas Y; D'Esposito, Mark

    2005-09-01

    We investigated the voluntary control of motor behavior by studying the process of deciding whether or not to execute a movement. We imaged the human dorsal cortex while subjects performed a countermanding task that allowed us to manipulate the probability that subjects would be able to cancel a planned saccade in response to an imperative stop signal. We modeled the behavioral data as a race between gaze-shifting mechanisms and gaze-holding mechanisms towards a finish line where a saccade is generated or canceled, and estimated that saccade cancelation took approximately 160 ms. The frontal eye fields showed greater activation on stop signal trials regardless of successful cancelation, suggesting coactivation of saccade and fixation mechanisms. The supplementary eye fields, however, distinguished between successful and unsuccessful cancelation, suggesting a role in monitoring performance. These oculomotor regions play distinct roles in the decision processes mediating saccadic choice. PMID:15616130

  15. [Study of residual levels of delayed-action neuroleptics].

    PubMed

    Furet, Y; Breteau, M; Etienne, T

    1991-01-01

    Pharmacokinetics of the depot antipsychotics are unclear and mainly depend on releasing from the depot site (according to a "flip-flop" model). Few data are available on residual plasma concentrations of those drugs. We have practiced 38 blood determinations among 15 patients treated by long-acting neuroleptics (10 by fluphenazine decanoate, 4 by flupentixol decanoate and 1 by pipotiazine palmitate). Radio Receptor Assay method was used (based on competition for dopamine receptors binding), with results expressed as chlorpromazine equivalents. They showed; a wide interindividual variability; considering each subject, intraindividual variability is attenuated; blood measurements are mainly higher than therapeutic ranges (especially for patients on fluphenazine decanoate). Those results might involve that some patients are overdosed, but other studies are needed in this way. PMID:1675816

  16. Study of the mode of action of some nitrodiphenyl ethers.

    PubMed

    Piekarski, D; Potier, P; Giannotti, C

    1990-01-01

    Nitrosoderivatives of the nitrodiphenyl ether herbicides (nitrofen, bifenox) have been studied. UV irradiation in different organic solvents gives degradation products. In buffered aqueous media, in the presence of chloroplasts and spin traps such as DMPO, hydroxy and peroxy radicals have been characterized. In organic media and in the presence of spin traps such as DMPO, PBN, 4-POBN, solvent radicals (.CHCl2, .CCl3, .CH2 [symbol: see text]) have been formed. Nitro-derivatives have been studied under UV irradiation and in the presence of tetramethylethylene (TME), alkenylhydroxylamines are formed which autoxidize in nitroxide radicals. The formation of the stable nitroxide radical occurs in the dark process after continuous irradiation. The intensity of the signal decreases strongly when a new irradiation is applied. Radical species, with analogous ESR spectral characteristics are formed on reaction with nitrodiphenyl ethers and fatty acids. The reactivity of these herbicides in micellar media (SDS, Brij 35, and CTAB) has been investigated. The kinetics of formation of the ESR signal corresponding to the photoreduction of the nitrodiphenyl ether in the presence of TME behave differently in a micellar environment as compared to solution. The intensity of the formation of the nitroxide increases under irradiation and decreases in the dark; the rotational correlation time tau c has been determined for each type of micelle. Synthetic nitrosodiphenyl ether made by the reduction of nitrodiphenyl ether using hydrogen gas and PtO2 as a catalyst gives the corresponding amine, which is oxidized with meta-chloroperbenzoic acid (m.CPBA). The nitrosodiphenyl ether in the presence of soja azolectin liposome containing a fluorescent probe has been analysed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2165990

  17. CRISPR/Cas9-mediated genome editing and gene replacement in plants: Transitioning from lab to field.

    PubMed

    Schaeffer, Scott M; Nakata, Paul A

    2015-11-01

    The CRISPR/Cas9 genome engineering system has ignited and swept through the scientific community like wildfire. Owing largely to its efficiency, specificity, and flexibility, the CRISPR/Cas9 system has quickly become the preferred genome-editing tool of plant scientists. In plants, much of the early CRISPR/Cas9 work has been limited to proof of concept and functional studies in model systems. These studies, along with those in other fields of biology, have led to the development of several utilities of CRISPR/Cas9 beyond single gene editing. Such utilities include multiplexing for inducing multiple cleavage events, controlling gene expression, and site specific transgene insertion. With much of the conceptual CRISPR/Cas9 work nearly complete, plant researchers are beginning to apply this gene editing technology for crop trait improvement. Before rational strategies can be designed to implement this technology to engineer a wide array of crops there is a need to expand the availability of crop-specific vectors, genome resources, and transformation protocols. We anticipate that these challenges will be met along with the continued evolution of the CRISPR/Cas9 system particularly in the areas of manipulation of large genomic regions, transgene-free genetic modification, development of breeding resources, discovery of gene function, and improvements upon CRISPR/Cas9 components. The CRISPR/Cas9 editing system appears poised to transform crop trait improvement. PMID:26475194

  18. Infants' perception of actions and situational constraints: an eye-tracking study.

    PubMed

    Elsner, Birgit; Pfeifer, Caroline; Parker, Charlene; Hauf, Petra

    2013-10-01

    Rational action understanding requires that infants evaluate the efficiency of a movement in achieving a goal with respect to situational constraints. In contrast, recent accounts have highlighted the impact of perceptual characteristics of the demonstrated movement or constraints to explain infants' behavior in so-called rational imitation tasks. The current study employed eye tracking to investigate how 13- to 15-month-old infants distribute their visual attention to different aspects of an action demonstration. In three tasks (touchlight, house, and obstacle), infants watched videos in which a model performed an unusual action while she was or was not restricted by situational constraints. Infants' overall looking to the demonstration as well as looking to four segments of the video (initial segment, constraint demonstration, action performance, and final segment) and to specific areas (constraint area of interest [AOI] and action AOI) was analyzed. Overall, infants looked longer at the demonstration in the constraint condition compared with the no-constraint condition. The condition differences occurred in the two video segments where the constraint or action was displayed and were especially profound for the constraint AOI. These findings indicate that infants processed the situational constraints. However, the pattern of condition differences varied slightly in the three tasks. In sum, the data imply that infants process perceptual characteristics of the movement or constraints and that low-level perceptual processes interact with higher level cognitive processes in infants' action perception. PMID:23410481

  19. Biosphere reserves in action: Case studies of the American experience

    SciTech Connect

    1995-06-26

    For nearly 20 years, biosphere reserves have offered a unique framework for building the knowledge, skills, and attitudes required for conservation and sustainable use of ecosystems. The 12 case studies in this volume chronicle many of the cooperative efforts to implement the biosphere reserve concept in the United States. Considered together, these efforts involve more than 20 types of protected areas, and the participation of all levels of government, and many private organizations, academic institutions, citizens groups, and individuals. Biosphere reserves are multi-purpose areas that are nominated by the national committee of the Man and the Biosphere Program (MAB) and designated by the United Nations Educational, Scientific and Cultural Organization (UNESCO) to serve as demonstration areas for cooperation in building harmonious relationships between human activities and the conservation of ecosystems and biological diversity. Each biosphere reserve exemplifies the characteristic ecosystems of one of the worlds biogeographical regions. It is a land or coas%arine area involving human communities as integral components and including resources managed for objectives ranging from complete protection to intensive, yet sustainable development. A biosphere reserve is envisioned as a regional ''landscape for learning'' in which monitoring, research, education, and training are encouraged to support sustainable conservation of natural and managed ecosystems. It is a framework for regional cooperation involving government decisionmakers, scientists, resource managers, private organizations and local people (i.e., the biosphere reserve ''stakeholders''). Finally, each biosphere reserve is part of a global network for sharing information and experience to help address complex problems of conservation and development. The 12 case studies presented in this report represent only a few of the possible evolutions of a biosphere reserve in its efforts to reach out to the local

  20. Streamlined Approach for Environmental Restoration Plan for Corrective Action Unit 330: Areas 6, 22, and 23 Tanks and Spill Sites, Nevada Test Site, Nevada

    SciTech Connect

    T. M. Fitzmaurice

    2001-08-01

    This Streamlined Approach for Environmental restoration (SAFER) plan addresses the action necessary for the closure of Corrective Action Unit (CAU) 330, Areas 6,22, and 23 Tanks and Spill Sites. The CAUs are currently listed in Appendix III of the Federal Facility Agreement and Consent Order (FFACO). This CAU is located at the Nevada Test Site (NTS) (Figure 1). CAU 330 consists of the following Corrective Action Sites (CASs): (1) CAS 06-02-04 - Consists of an underground tank and piping. This CAS is close to an area that was part of the Animal Investigation Program (AIP), conducted under the U.S. Public Health Service. Its purpose was to study and perform tests on the cattle and wild animals in and around the NTS that were exposed to radionuclides. It is unknown if this tank was part of these operations. (2) CAS 22-99-06 - Is a fuel spill that is believed to be a waste oil release which occurred when Camp Desert Rock was an active facility. This CAS was originally identified as being a small depression where liquids were poured onto the ground, located on the west side of Building T-1001. This building has been identified as housing a fire station, radio station, and radio net remote and telephone switchboard. (3) CAS 23-01-02 - Is a large aboveground storage tank (AST) farm that was constructed to provide gasoline and diesel storage in Area 23. The site consists of two ASTs, a concrete foundation, a surrounding earthen berm, associated piping, and unloading stations. (4) CAS 23-25-05 - Consists of an asphalt oil spill/tar release that contains a wash covered with asphalt oil/tar material, a half buried 208-liter (L) (55-gallon [gal]) drum, rebar, and concrete located in the vicinity.

  1. CLOSURE REPORT FOR CORRECTIVE ACTION UNIT 204: STORAGE BUNKERS, NEVADA TEST SITE, NEVADA

    SciTech Connect

    2006-04-01

    Corrective Action Unit (CAU) 330 consists of four Corrective Action Sites (CASs) located in Areas 6, 22, and 23 of the Nevada Test Site (NTS). The unit is listed in the Federal Facility Agreement and Consent Order (FFACO, 1996) as CAU 330: Areas 6, 22, and 23 Tanks and Spill Sites. CAU 330 consists of the following CASs: CAS 06-02-04, Underground Storage Tank (UST) and Piping CAS 22-99-06, Fuel Spill CAS 23-01-02, Large Aboveground Storage Tank (AST) Farm CAS 23-25-05, Asphalt Oil Spill/Tar Release

  2. Closure Report for Housekeeping Category Corrective Action Unit 345 Nevada Test Site, Nevada

    SciTech Connect

    A. T. Urbon

    2000-11-01

    This Closure Report for Corrective Action Unit (CAU) 345 summarizes the disposition of ten Corrective Action Sites (CAS) located in Areas 2 and 9 of the Nevada Test Site, Nevada. The table listed in the report provides a description of each CAS and the status of its associated waste as listed in the ''Federal Facilities Agreement and Consent Order'' (FFACO, 1996). Copies of the Sectored Housekeeping Site Closure Verification Form for each CAS are included as Attachment A. The battery at CAS 09-24-04 required sampling for waste disposal purposes. The waste was found to be not hazardous. Results of the sampling are included in Attachment B.

  3. Some studies on the rodenticidal action of indomethacin.

    PubMed

    Omogbai, E K; Ozolua, R I; Idaewor, P E; Isah, A O

    1999-11-01

    The oral LD50 of indomethacin for a seven-day observation was found to be 12.58 +/- 1.15 mg/kg. At LD10 of 6.61 mg/kg, a dose to weight ratio of 28 was obtained for a 240 g rat, while at a maximum single dose of 3 mg/kg in man it is only 0.04. Neither diazepam nor phenobarbital influenced death at the doses of both drugs used. However, cholestyramine 2 g/kg/day was found to protect by 50% from the LD100 of indomethacin. Gross pathological studies showed dose-dependent ulceration and perforation (P < 0.001, 12 vs 24 mg/kg) and such lesions occurred in starved rats, were low in bile duct-ligated compared to sham-operated rats (P < 0.001) and were also low in cholestyramine-treated rats. Indomethacin-induced lethality in rats was found to be dose-dependent. PMID:10536753

  4. Corrective Action Plan for Corrective Action Unit 563: Septic Systems, Nevada Test Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2009-03-31

    This Corrective Action Plan (CAP) has been prepared for Corrective Action Unit (CAU) 563, Septic Systems, in accordance with the Federal Facility Agreement and Consent Order. CAU 563 consists of four Corrective Action Sites (CASs) located in Areas 3 and 12 of the Nevada Test Site. CAU 563 consists of the following CASs: CAS 03-04-02, Area 3 Subdock Septic Tank CAS 03-59-05, Area 3 Subdock Cesspool CAS 12-59-01, Drilling/Welding Shop Septic Tanks CAS 12-60-01, Drilling/Welding Shop Outfalls Site characterization activities were performed in 2007, and the results are presented in Appendix A of the CAU 563 Corrective Action Decision Document. The scope of work required to implement the recommended closure alternatives is summarized below. CAS 03-04-02, Area 3 Subdock Septic Tank, contains no contaminants of concern (COCs) above action levels. No further action is required for this site; however, as a best management practice (BMP), all aboveground features (e.g., riser pipes and bumper posts) will be removed, the septic tank will be removed, and all open pipe ends will be sealed with grout. CAS 03-59-05, Area 3 Subdock Cesspool, contains no COCs above action levels. No further action is required for this site; however, as a BMP, all aboveground features (e.g., riser pipes and bumper posts) will be removed, the cesspool will be abandoned by filling it with sand or native soil, and all open pipe ends will be sealed with grout. CAS 12-59-01, Drilling/Welding Shop Septic Tanks, will be clean closed by excavating approximately 4 cubic yards (yd3) of arsenic- and chromium-impacted soil. In addition, as a BMP, the liquid in the South Tank will be removed, the North Tank will be removed or filled with grout and left in place, the South Tank will be filled with grout and left in place, all open pipe ends will be sealed with grout or similar material, approximately 10 yd3 of chlordane-impacted soil will be excavated, and debris within the CAS boundary will be removed. CAS 12

  5. The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA complex

    SciTech Connect

    Wang, Ruiying; Zheng, Han; Preamplume, Gan; Shao, Yaming; Li, Hong

    2012-03-15

    The repeat-associated mysterious proteins (RAMPs) comprise the most abundant family of proteins involved in prokaryotic immunity against invading genetic elements conferred by the clustered regularly interspaced short palindromic repeat (CRISPR) system. Cas6 is one of the first characterized RAMP proteins and is a key enzyme required for CRISPR RNA maturation. Despite a strong structural homology with other RAMP proteins that bind hairpin RNA, Cas6 distinctly recognizes single-stranded RNA. Previous structural and biochemical studies show that Cas6 captures the 5' end while cleaving the 3' end of the CRISPR RNA. Here, we describe three structures and complementary biochemical analysis of a noncatalytic Cas6 homolog from Pyrococcus horikoshii bound to CRISPR repeat RNA of different sequences. Our study confirms the specificity of the Cas6 protein for single-stranded RNA and further reveals the importance of the bases at Positions 5-7 in Cas6-RNA interactions. Substitutions of these bases result in structural changes in the protein-RNA complex including its oligomerization state.

  6. CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots.

    PubMed

    Cai, Yupeng; Chen, Li; Liu, Xiujie; Sun, Shi; Wu, Cunxiang; Jiang, Bingjun; Han, Tianfu; Hou, Wensheng

    2015-01-01

    As a new technology for gene editing, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been rapidly and widely used for genome engineering in various organisms. In the present study, we successfully applied type II CRISPR/Cas9 system to generate and estimate genome editing in the desired target genes in soybean (Glycine max (L.) Merrill.). The single-guide RNA (sgRNA) and Cas9 cassettes were assembled on one vector to improve transformation efficiency, and we designed a sgRNA that targeted a transgene (bar) and six sgRNAs that targeted different sites of two endogenous soybean genes (GmFEI2 and GmSHR). The targeted DNA mutations were detected in soybean hairy roots. The results demonstrated that this customized CRISPR/Cas9 system shared the same efficiency for both endogenous and exogenous genes in soybean hairy roots. We also performed experiments to detect the potential of CRISPR/Cas9 system to simultaneously edit two endogenous soybean genes using only one customized sgRNA. Overall, generating and detecting the CRISPR/Cas9-mediated genome modifications in target genes of soybean hairy roots could rapidly assess the efficiency of each target loci. The target sites with higher efficiencies can be used for regular soybean transformation. Furthermore, this method provides a powerful tool for root-specific functional genomics studies in soybean. PMID:26284791

  7. 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. PMID:26673779

  8. Efficiency and Inheritance of Targeted Mutagenesis in Maize Using CRISPR-Cas9.

    PubMed

    Zhu, Jinjie; Song, Ning; Sun, Silong; Yang, Weilong; Zhao, Haiming; Song, Weibin; Lai, Jinsheng

    2016-01-20

    CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently, it has been developed as a powerful targeted genome editing tool for a wide variety of species. However, its application in maize has only been tested with transiently expressed somatic cells or with a limited number of stable transgenic T0 plants. The exact efficiency and specificity of the CRISPR/Cas system in the highly complex maize genome has not been documented yet. Here we report an extensive study of the well-studied type II CRISPR-Cas9 system for targeted genome editing in maize, with the codon-optimized Cas9 protein and the short non-coding guide RNA generated through a functional maize U6 snRNA promoter. Targeted gene mutagenesis was detected for 90 loci by maize protoplast assay, with an average cleavage efficiency of 10.67%. Stable knockout transformants for maize phytoene synthase gene (PSY1) were obtained. Mutations occurred in germ cells can be stably inherited to the next generation. Moreover, no off-target effect was detected at the computationally predicted putative off-target loci. No significant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was detected. Our results confirmed that the CRISPR-Cas9 could be successfully applied as a robust targeted genome editing system in maize. PMID:26842991

  9. Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system.

    PubMed

    Gao, Shuliang; Tong, Yangyang; Wen, Zhiqiang; Zhu, Li; Ge, Mei; Chen, Daijie; Jiang, Yu; Yang, Sheng

    2016-08-01

    Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica. PMID:27349768

  10. Foreign DNA acquisition by the I-F CRISPR–Cas system requires all components of the interference machinery

    PubMed Central

    Vorontsova, Daria; Datsenko, Kirill A.; Medvedeva, Sofia; Bondy-Denomy, Joseph; Savitskaya, Ekaterina E.; Pougach, Ksenia; Logacheva, Maria; Wiedenheft, Blake; Davidson, Alan R.; Severinov, Konstantin; Semenova, Ekaterina

    2015-01-01

    CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR–Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR–Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR–Cas systems. PMID:26586803

  11. A 'suicide' CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans.

    PubMed

    Wang, Yu; Wei, Dongsheng; Zhu, Xiangyang; Pan, Jiao; Zhang, Ping; Huo, Liang; Zhu, Xudong

    2016-01-01

    Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This 'suicide' CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans. PMID:27503169

  12. A ‘suicide’ CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans

    PubMed Central

    Wang, Yu; Wei, Dongsheng; Zhu, Xiangyang; Pan, Jiao; Zhang, Ping; Huo, Liang; Zhu, Xudong

    2016-01-01

    Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This ‘suicide’ CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans. PMID:27503169

  13. [A comparison of the knockout efficiencies of two codon-optimized Cas9 coding sequences in zebrafish embryos].

    PubMed

    Fenghua, Zhang; Houpeng, Wang; Siyu, Huang; Feng, Xiong; Zuoyan, Zhu; Yonghua, Sun

    2016-02-01

    Recent years have witnessed the rapid development of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas9)system. In order to realize gene knockout with high efficiency and specificity in zebrafish, several labs have synthesized distinct Cas9 cDNA sequences which were cloned into different vectors. In this study, we chose two commonly used zebrafish-codon-optimized Cas9 coding sequences (zCas9_bz, zCas9_wc) from two different labs, and utilized them to knockout seven genes in zebrafish embryos, including the exogenous egfp and six endogenous genes (chd, hbegfa, th, eef1a1b, tyr and tcf7l1a). We compared the knockout efficiencies resulting from the two zCas9 coding sequences, by direct sequencing of PCR products, colony sequencing and phenotypic analysis. The results showed that the knockout efficiency of zCas9_wc was higher than that of zCas9_bz in all conditions. PMID:26907778

  14. Corrective Action Plan for Corrective Action Unit 166: Storage Yards and Contaminated Materials, Nevada Test Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2007-10-01

    Corrective Action Unit (CAU) 166, Storage Yards and Contaminated Materials, is listed in the Federal Facility Agreement and Consent Order (FFACO) of 1996 (FFACO, 1996). CAU 166 consists of seven Corrective Action Sites (CASs) located in Areas 2, 3, 5, and 18 of the Nevada Test Site (NTS), which is located approximately 65 miles northwest of Las Vegas, Nevada (Figure 1). CAU 166 consists of the following CASs: (1) CAS 02-42-01, Cond. Release Storage Yd - North; (2) CAS 02-42-02, Cond. Release Storage Yd - South; (3) CAS 02-99-10, D-38 Storage Area; (4) CAS 03-42-01, Conditional Release Storage Yard; (5) CAS 05-19-02, Contaminated Soil and Drum; (6) CAS 18-01-01, Aboveground Storage Tank; and (7) CAS 18-99-03, Wax Piles/Oil Stain. Details of the site history and site characterization results for CAU 166 are provided in the approved Corrective Action Investigation Plan (CAIP) (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2006) and in the approved Corrective Action Decision Document (CADD) (NNSA/NSO, 2007).

  15. Translational studies of goal-directed action as a framework for classifying deficits across psychiatric disorders

    PubMed Central

    Griffiths, Kristi R.; Morris, Richard W.; Balleine, Bernard W.

    2014-01-01

    The ability to learn contingencies between actions and outcomes in a dynamic environment is critical for flexible, adaptive behavior. Goal-directed actions adapt to changes in action-outcome contingencies as well as to changes in the reward-value of the outcome. When networks involved in reward processing and contingency learning are maladaptive, this fundamental ability can be lost, with detrimental consequences for decision-making. Impaired decision-making is a core feature in a number of psychiatric disorders, ranging from depression to schizophrenia. The argument can be developed, therefore, that seemingly disparate symptoms across psychiatric disorders can be explained by dysfunction within common decision-making circuitry. From this perspective, gaining a better understanding of the neural processes involved in goal-directed action, will allow a comparison of deficits observed across traditional diagnostic boundaries within a unified theoretical framework. This review describes the key processes and neural circuits involved in goal-directed decision-making using evidence from animal studies and human neuroimaging. Select studies are discussed to outline what we currently know about causal judgments regarding actions and their consequences, action-related reward evaluation, and, most importantly, how these processes are integrated in goal-directed learning and performance. Finally, we look at how adaptive decision-making is impaired across a range of psychiatric disorders and how deepening our understanding of this circuitry may offer insights into phenotypes and more targeted interventions. PMID:24904322

  16. Motor Inhibition during Overt and Covert Actions: An Electrical Neuroimaging Study

    PubMed Central

    Angelini, Monica; Calbi, Marta; Ferrari, Annachiara; Sbriscia-Fioretti, Beatrice; Franca, Michele; Gallese, Vittorio; Umiltà, Maria Alessandra

    2015-01-01

    Given ample evidence for shared cortical structures involved in encoding actions, whether or not subsequently executed, a still unsolved problem is the identification of neural mechanisms of motor inhibition, preventing “covert actions” as motor imagery from being performed, in spite of the activation of the motor system. The principal aims of the present study were the evaluation of: 1) the presence in covert actions as motor imagery of putative motor inhibitory mechanisms; 2) their underlying cerebral sources; 3) their differences or similarities with respect to cerebral networks underpinning the inhibition of overt actions during a Go/NoGo task. For these purposes, we performed a high density EEG study evaluating the cerebral microstates and their related sources elicited during two types of Go/NoGo tasks, requiring the execution or withholding of an overt or a covert imagined action, respectively. Our results show for the first time the engagement during motor imagery of key nodes of a putative inhibitory network (including pre-supplementary motor area and right inferior frontal gyrus) partially overlapping with those activated for the inhibition of an overt action during the overt NoGo condition. At the same time, different patterns of temporal recruitment in these shared neural inhibitory substrates are shown, in accord with the intended overt or covert modality of action performance. The evidence that apparently divergent mechanisms such as controlled inhibition of overt actions and contingent automatic inhibition of covert actions do indeed share partially overlapping neural substrates, further challenges the rigid dichotomy between conscious, explicit, flexible and unconscious, implicit, inflexible forms of motor behavioral control. PMID:26000451

  17. Streamlined Approach for Environmental Restoration Plan for Corrective Action Unit 107: Low Impact Soil Sites, Nevada Test Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2009-03-31

    This Streamlined Approach for Environmental Restoration Plan covers activities associated with Corrective Action Unit (CAU) 107 of the Federal Facility Agreement and Consent Order (1996 [as amended February 2008]). CAU 107 consists of the following Corrective Action Sites (CASs) located in Areas 1, 2, 3, 4, 5, 9, 10, and 18 of the Nevada Test Site. {sm_bullet} CAS 01-23-02, Atmospheric Test Site - High Alt{sm_bullet} CAS 02-23-02, Contaminated Areas (2){sm_bullet} CAS 02-23-03, Contaminated Berm{sm_bullet} CAS 02-23-10, Gourd-Amber Contamination Area{sm_bullet} CAS 02-23-11, Sappho Contamination Area{sm_bullet} CAS 02-23-12, Scuttle Contamination Area{sm_bullet} CAS 03-23-24, Seaweed B Contamination Area{sm_bullet} CAS 03-23-27, Adze Contamination Area{sm_bullet} CAS 03-23-28, Manzanas Contamination Area{sm_bullet} CAS 03-23-29, Truchas-Chamisal Contamination Area{sm_bullet} CAS 04-23-02, Atmospheric Test Site T4-a{sm_bullet} CAS 05-23-06, Atmospheric Test Site{sm_bullet} CAS 09-23-06, Mound of Contaminated Soil{sm_bullet} CAS 10-23-04, Atmospheric Test Site M-10{sm_bullet} CAS 18-23-02, U-18d Crater (Sulky) Based on historical documentation, personnel interviews, site process knowledge, site visits, photographs, engineering drawings, field screening, analytical results, and the results of data quality objectives process (Section 3.0), closure in place with administrative controls or no further action will be implemented for CAU 107.

  18. Regulation of the Type I-F CRISPR-Cas system by CRP-cAMP and GalM controls spacer acquisition and interference

    PubMed Central

    Patterson, Adrian G.; Chang, James T.; Taylor, Corinda; Fineran, Peter C.

    2015-01-01

    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. PMID:26007654

  19. Expression of OsCAS (Calcium-Sensing Receptor) in an Arabidopsis Mutant Increases Drought Tolerance

    PubMed Central

    Wei, Rongrong; Liu, Yang

    2015-01-01

    The calcium-sensing receptor (CaS), which is localized in the chloroplasts, is a crucial regulator of extracellular calcium-induced stomatal closure in Arabidopsis. It has homologs in Oryza sativa and other plants. These sequences all have a rhodanese-like protein domain, which has been demonstrated to be associated with specific stress conditions. In this study, we cloned the Oryza sativa calcium-sensing receptor gene (OsCAS) and demonstrated that OsCAS could sense an increase of extracellular Ca2+ concentration and mediate an increase in cytosolic Ca2+ concentration. The OsCAS gene was transformed into an Arabidopsis CaS knockout mutant (Salk) and overexpressed in the transgenic plants. OsCAS promoted stomatal closure. We screened homozygous transgenic Arabidopsis plants and determined physiological indices such as the oxidative damage biomarker malondialdehyde (MDA), relative membrane permeability (RMP), proline content, and chlorophyll fluorescence parameters, after 21 days of drought treatment. Our results revealed lower RMP and MDA contents and a higher Proline content in transgenic Arabidopsis plants after drought stress, whereas the opposite was observed in Salk plants. With respect to chlorophyll fluorescence, the electron transport rate and effective PSII quantum yield decreased in all lines under drought stress; however, in the transgenic plants these two parameters changed fewer and were higher than those in wild-type and Salk plants. The quantum yield of regulated energy dissipation and nonregulated energy dissipation in PSII were higher in Salk plants, whereas these values were lower in the transgenic plants than in the wild type under drought stress. The above results suggest that the transgenic plants showed better resistance to drought stress by decreasing damage to the cell membrane, increasing the amount of osmoprotectants, and maintaining a relatively high photosynthetic capacity. In conclusion, OsCAS is an extracellular calcium-sensing receptor

  20. CRISPR-Cas Adaptive Immune Systems of the Sulfolobales: Unravelling Their Complexity and Diversity

    PubMed Central

    Garrett, Roger A.; Shah, Shiraz A.; Erdmann, Susanne; Liu, Guannan; Mousaei, Marzieh; León-Sobrino, Carlos; Peng, Wenfang; Gudbergsdottir, Soley; Deng, Ling; Vestergaard, Gisle; Peng, Xu; She, Qunxin

    2015-01-01

    The Sulfolobales have provided good model organisms for studying CRISPR-Cas systems of the crenarchaeal kingdom of the archaea. These organisms are infected by a wide range of exceptional archaea-specific viruses and conjugative plasmids, and their CRISPR-Cas systems generally exhibit extensive structural and functional diversity. They carry large and multiple CRISPR loci and often multiple copies of diverse Type I and Type III interference modules as well as more homogeneous adaptation modules. These acidothermophilic organisms have recently provided seminal insights into both the adaptation process, the diverse modes of interference, and their modes of regulation. The functions of the adaptation and interference modules tend to be loosely coupled and the stringency of the crRNA-DNA sequence matching during DNA interference is relatively low, in contrast to some more streamlined CRISPR-Cas systems of bacteria. Despite this, there is evidence for a complex and differential regulation of expression of the diverse functional modules in response to viral infection. Recent work also supports critical roles for non-core Cas proteins, especially during Type III-directed interference, and this is consistent with these proteins tending to coevolve with core Cas proteins. Various novel aspects of CRISPR-Cas systems of the Sulfolobales are considered including an alternative spacer acquisition mechanism, reversible spacer acquisition, the formation and significance of antisense CRISPR RNAs, and a novel mechanism for avoidance of CRISPR-Cas defense. Finally, questions regarding the basis for the complexity, diversity, and apparent redundancy, of the intracellular CRISPR-Cas systems are discussed. PMID:25764276

  1. New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.

    PubMed

    Laughery, Marian F; Hunter, Tierra; Brown, Alexander; Hoopes, James; Ostbye, Travis; Shumaker, Taven; Wyrick, John J

    2015-12-01

    Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology is an important tool for genome editing because the Cas9 endonuclease can induce targeted DNA double-strand breaks. Targeting of the DNA break is typically controlled by a single-guide RNA (sgRNA), a chimeric RNA containing a structural segment important for Cas9 binding and a 20mer guide sequence that hybridizes to the genomic DNA target. Previous studies have demonstrated that CRISPR-Cas9 technology can be used for efficient, marker-free genome editing in Saccharomyces cerevisiae. However, introducing the 20mer guide sequence into yeast sgRNA expression vectors often requires cloning procedures that are complex, time-consuming and/or expensive. To simplify this process, we have developed a new sgRNA expression cassette with internal restriction enzyme sites that permit rapid, directional cloning of 20mer guide sequences. Here we describe a flexible set of vectors based on this design for cloning and expressing sgRNAs (and Cas9) in yeast using different selectable markers. We anticipate that the Cas9-sgRNA expression vector with the URA3 selectable marker (pML104) will be particularly useful for genome editing in yeast, since the Cas9 machinery can be easily removed by counter-selection using 5-fluoro-orotic acid (5-FOA) following successful genome editing. The availability of new vectors that simplify and streamline the technical steps required for guide sequence cloning should help accelerate the use of CRISPR-Cas9 technology in yeast genome editing. PMID:26305040

  2. Expression of OsCAS (Calcium-Sensing Receptor) in an Arabidopsis Mutant Increases Drought Tolerance.

    PubMed

    Zhao, Xin; Xu, Mengmeng; Wei, Rongrong; Liu, Yang

    2015-01-01

    The calcium-sensing receptor (CaS), which is localized in the chloroplasts, is a crucial regulator of extracellular calcium-induced stomatal closure in Arabidopsis. It has homologs in Oryza sativa and other plants. These sequences all have a rhodanese-like protein domain, which has been demonstrated to be associated with specific stress conditions. In this study, we cloned the Oryza sativa calcium-sensing receptor gene (OsCAS) and demonstrated that OsCAS could sense an increase of extracellular Ca2+ concentration and mediate an increase in cytosolic Ca2+ concentration. The OsCAS gene was transformed into an Arabidopsis CaS knockout mutant (Salk) and overexpressed in the transgenic plants. OsCAS promoted stomatal closure. We screened homozygous transgenic Arabidopsis plants and determined physiological indices such as the oxidative damage biomarker malondialdehyde (MDA), relative membrane permeability (RMP), proline content, and chlorophyll fluorescence parameters, after 21 days of drought treatment. Our results revealed lower RMP and MDA contents and a higher Proline content in transgenic Arabidopsis plants after drought stress, whereas the opposite was observed in Salk plants. With respect to chlorophyll fluorescence, the electron transport rate and effective PSII quantum yield decreased in all lines under drought stress; however, in the transgenic plants these two parameters changed fewer and were higher than those in wild-type and Salk plants. The quantum yield of regulated energy dissipation and nonregulated energy dissipation in PSII were higher in Salk plants, whereas these values were lower in the transgenic plants than in the wild type under drought stress. The above results suggest that the transgenic plants showed better resistance to drought stress by decreasing damage to the cell membrane, increasing the amount of osmoprotectants, and maintaining a relatively high photosynthetic capacity. In conclusion, OsCAS is an extracellular calcium-sensing receptor

  3. Transforming Language Ideologies through Action Research: A Case Study of Bilingual Science Learning

    NASA Astrophysics Data System (ADS)

    Yang, Eunah

    This qualitative case study explored a third grade bilingual teacher's transformative language ideologies through participating in a collaborative action research project. By merging language ideologies theory, Cultural Historical Activity Theory (CHAT), and action research, I was able to identify the analytic focus of this study. I analyzed how one teacher and I, the researcher, collaboratively reflected on classroom language practices during the video analysis meetings and focus groups. Further, I analyzed twelve videos that we coded together to see the changes in the teacher's language practices over time. My unit of analysis was the discourse practice mediated by additive language ideologies. Throughout the collaborative action research process, we both critically reflected on the classroom language use. We also developed a critical consciousness about the participatory shifts and learning of focal English Learner (EL) students. Finally, the teacher made changes to her classroom language practices. The results of this study will contribute to the literacy education research field for theoretical, methodological, and practical insights. The integration of language ideologies, CHAT, and action research can help educational practitioners, researchers, and policy makers understand the importance of transforming teachers' language ideologies in designing additive learning contexts for ELs. From a methodological perspective, the transformative language ideologies through researcher and teacher collaborated video analysis process provide a unique contribution to the language ideologies in education literature, with analytic triangulation. As a practical implication, this study suggests action research can be one of the teacher education tools to help the teachers transform language ideologies for EL education.

  4. Anatomical correlates for category-specific naming of objects and actions: a brain stimulation mapping study.

    PubMed

    Lubrano, Vincent; Filleron, Thomas; Démonet, Jean-François; Roux, Franck-Emmanuel

    2014-02-01

    The production of object and action words can be dissociated in aphasics, yet their anatomical correlates have been difficult to distinguish in functional imaging studies. To investigate the extent to which the cortical neural networks underlying object- and action-naming processing overlap, we performed electrostimulation mapping (ESM), which is a neurosurgical mapping technique routinely used to examine language function during brain-tumor resections. Forty-one right-handed patients who had surgery for a brain tumor were asked to perform overt naming of object and action pictures under stimulation. Overall, 73 out of the 633 stimulated cortical sites (11.5%) were associated with stimulation-induced language interferences. These interference sites were very much localized (<1 cm(2) ), and showed substantial variability across individuals in their exact localization. Stimulation interfered with both object and action naming over 44 sites, whereas it specifically interfered with object naming over 19 sites and with action naming over 10 sites. Specific object-naming sites were mainly identified in Broca's area (Brodmann area 44/45) and the temporal cortex, whereas action-naming specific sites were mainly identified in the posterior midfrontal gyrus (Brodmann area 6/9) and Broca's area (P = 0.003 by the Fisher's exact test). The anatomical loci we emphasized are in line with a cortical distinction between objects and actions based on conceptual/semantic features, so the prefrontal/premotor cortex would preferentially support sensorimotor contingencies associated with actions, whereas the temporal cortex would preferentially underpin (functional) properties of objects. PMID:23015527

  5. Motions of alloying additions in the CAS steelmaking operations

    NASA Astrophysics Data System (ADS)

    Mazumdar, D.; Guthrie, R. I. L.

    1993-08-01

    Water model studies in a pilot scale ladle ( D = 1.12 m and L = 0.93 m) were carried out to investigate the subsurface motion of both buoyant and sinking additions during the CAS (com-position adjustment by sealed argon bubbling systems) alloy addition procedure in steelmaking. This technique involves placing a refractory baffle around a rising gas/liquid plume within a stirred ladle of steel. Alloy additions are then made by projecting them into the slag-free region of steel within the baffled region. It was found that such particles while moving through the upwelling two-phase plume region can experience a significant reduction in drag forces, causing buoyant particles to penetrate more deeply than anticipated for a homogeneous fluid. Therefore, considering reduced drag on particles penetrating the upwelling gas liquid plume region, predictions were made for the trajectories of spherical-shaped particles using Newton’s law of motion. Predictions were in very reasonable agreement with those measured. Incorporating the velocity fields in industrial size vessels already reported by the present authors, trajectories of spherical-shaped additions (diameter ˜ 80 mm) in a 150-ton ladle during CAS operations were then predicted. The industrial implications of such trajectories, together with the alloy’s dissolution and dispersion behavior, were also analyzed. Finally, advantages of the CAS alloy addition procedure over conventional methods, in terms of the recovery rates of buoyant additions, are discussed.

  6. Targeted mutagenesis using CRISPR/Cas system in medaka

    PubMed Central

    Ansai, Satoshi; Kinoshita, Masato

    2014-01-01

    ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-based RNA-guided endonuclease (RGEN) has recently emerged as a simple and efficient tool for targeted genome editing. In this study, we showed successful targeted mutagenesis using RGENs in medaka, Oryzias latipes. Somatic and heritable mutations were induced with high efficiency at the targeted genomic sequence on the DJ-1 gene in embryos that had been injected with the single guide RNA (sgRNA) transcribed by a T7 promoter and capped RNA encoding a Cas9 nuclease. The sgRNAs that were designed for the target genomic sequences without the 5′ end of GG required by the T7 promoter induced the targeted mutations. This suggests that the RGEN can target any sequence adjacent to an NGG protospacer adjacent motif (PAM) sequence, which occurs once every 8 bp. The off-target alterations at 2 genomic loci harboring double mismatches in the 18-bp targeting sequences were induced in the RGEN-injected embryos. However, we also found that the off-target effects could be reduced by lower dosages of sgRNA. Taken together, our results suggest that CRISPR/Cas-mediated RGENs may be an efficient and flexible tool for genome editing in medaka. PMID:24728957

  7. Cancer, Employment, and American Indians: A Participatory Action Research Pilot Study

    ERIC Educational Resources Information Center

    Johnson, Sharon R.; Finifrock, DeAnna; Marshall, Catherine A.; Jaakola, Julia; Setterquist, Janette; Burross, Heidi L.; Hodge, Felicia Schanche

    2011-01-01

    American Indian cancer survivors are an underserved and understudied group. In this pilot study we attempted to address, through participatory action research, missing information about those factors that serve to either facilitate employment or hinder it for adult cancer survivors. One task of the study was to develop and/or modify…

  8. Reconstructing a Community, Reclaiming a Playground: A Participatory Action Research Study

    ERIC Educational Resources Information Center

    Hutzel, Karen

    2007-01-01

    This article describes a participatory action research study that examined participant's perceptions of community and of the West End neighborhood in Cincinnati, Ohio, where the study took place. It is argued that oppressive situations have developed strong collective identities and social capital among residents, which can lead to the development…

  9. Understanding the Impact of Affirmative Action Bans in Different Graduate Fields of Study

    ERIC Educational Resources Information Center

    Garces, Liliana M.

    2013-01-01

    This study examines the effects of affirmative action bans in four states (California, Florida, Texas, and Washington) on the enrollment of underrepresented students of color within six different graduate fields of study: the natural sciences, engineering, social sciences, business, education, and humanities. Findings show that affirmative action…

  10. A Study of Participatory Action Research as Professional Development for Educators in Areas of Educational Disadvantage

    ERIC Educational Resources Information Center

    James, E. Alana

    2006-01-01

    This paper presents the final analysis of a mixed methodological study of participatory action research (PAR) as professional development. The participants were administrators and teachers studying extreme educational disadvantage caused by homeless and transient living conditions. Two questions are answered: 1. What was the experience of…

  11. Reconnecting with Your Passion: An Action Research Study Exploring Humanities and Professional Nursing

    ERIC Educational Resources Information Center

    Snyder, Melissa J.

    2012-01-01

    The purpose of this action research study was two-fold. The first purpose was to examine the process of how nurses engaged in a professional development program that drew upon reading and creative writing related to their lives and work as nurses. Secondly, this study examined the nurses' perspectives on how their involvement in the process…

  12. Changing Preschool Children's Attitudes into Behavior towards Selected Environmental Issues: An Action Research Study

    ERIC Educational Resources Information Center

    Ertürk Kara, Gözde; Aydos, E. Hande; Aydin, Özge

    2015-01-01

    The purpose of this study is to provide the transform of attitudes into behavior of 60-72 month of age children continued early childhood education toward environmental issues. Collaborative action research method of qualitative design was used. The whole participants of the study were 60-72 months of age children who were attending in an early…

  13. AND-34/BCAR3 Regulates Adhesion-Dependent p130Cas Serine Phosphorylation and Breast Cancer Cell Growth Pattern

    PubMed Central

    Makkinje, Anthony; Near, Richard I.; Infusini, Giuseppe; Borre, Pierre Vanden; Bloom, Alexander; Cai, Dongpo; Costello, Catherine E.; Lerner, Adam

    2009-01-01

    NSP protein family members associate with p130Cas, a focal adhesion adapter protein best known as a Src substrate that integrates adhesion-related signaling. Over-expression of AND-34/BCAR3/NSP2 (BCAR3), but not NSP1 or NSP3, induces anti-estrogen resistance in human breast cancer cell lines. BCAR3 over-expression in epithelial MCF-7 cells augments levels of a phosphorylated p130Cas species that migrates more slowly on SDS PAGE while NSP-1 and NSP3 induce modest or no phosphorylation, respectively. Conversely, reduction in BCAR3 expression in mesenchymal MDA-231 cells by inducible shRNA results in loss of such p130Cas phosphorylation. Replacement of NSP3's serine/proline-rich domain with that of AND-34/BCAR3 instills the ability to induce p130Cas phosphorylation. Phospho-amino acid analysis demonstrates that BCAR3 induces p130Cas serine phosphorylation. Mass spectrometry identified phosphorylation at p130Cas serines 139, 437 and 639. p130Cas serine phosphorylation accumulates for several hours after adhesion of MDA-231 cells to fibronectin and is dependent upon BCAR3 expression. BCAR3 knockdown alters p130Cas localization and converts MDA-231 growth to an epithelioid pattern characterized by striking cohesiveness and lack of cellular projections at colony borders. These studies demonstrate that BCAR3 regulates p130Cas serine phosphorylation that is adhesion-dependent, temporally distinct from previously well-characterized rapid Fak and Src kinase-mediated p130Cas tyrosine phosphorylation and that correlates with invasive phenotype. PMID:19454314

  14. Diversity of CRISPR-Cas-Mediated Mechanisms of Adaptive Immunity in Prokaryotes and Their Application in Biotechnology.

    PubMed

    Savitskaya, E E; Musharova, O S; Severinov, K V

    2016-07-01

    CRISPR-Cas systems of adaptive immunity in prokaryotes consist of CRISPR arrays (clusters of short repeated genomic DNA fragments separated by unique spacer sequences) and cas (CRISPR-associated) genes that provide cells with resistance against bacteriophages and plasmids containing protospacers, i.e. sequences complementary to CRISPR array spacers. CRISPR-Cas systems are responsible for two different cellular phenomena: CRISPR adaptation and CRISPR interference. CRISPR adaptation is cell genome modification by integration of new spacers that represents a unique case of Lamarckian inheritance. CRISPR interference involves specific recognition of protospacers in foreign DNA followed by introduction of breaks into this DNA and its destruction. According to the mechanisms of action, CRISPR-Cas systems have been subdivided into two classes, five types, and numerous subtypes. The development of techniques based on CRISPR interference mediated by the Type II system Cas9 protein has revolutionized the field of genome editing because it allows selective, efficient, and relatively simple introduction of directed breaks into target DNA loci. However, practical applications of CRISPR-Cas systems are not limited only to genome editing. In this review, we focus on the variety of CRISPR interference and CRISPR adaptation mechanisms and their prospective use in biotechnology. PMID:27449612

  15. Corrective Action Plan for Corrective Action Unit 543: Liquid Disposal Units, Nevada Test Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2007-04-01

    Corrective Action Unit (CAU) 543: Liquid Disposal Units is listed in Appendix III of the ''Federal Facility Agreement and Consent Order'' (FFACO) which was agreed to by the state of Nevada, the U.S. Department of Energy (DOE), and the U.S. Department of Defense (FFACO, 1996). CAU 543 sites are located in Areas 6 and 15 of the Nevada Test Site (NTS), which is approximately 65 miles northwest of Las Vegas, Nevada. CAU 543 consists of the following seven Corrective Action Sites (CASs) (Figure 1): CAS 06-07-01, Decon Pad; CAS 15-01-03, Aboveground Storage Tank; CAS 15-04-01, Septic Tank; CAS 15-05-01, Leachfield; CAS 15-08-01, Liquid Manure Tank; CAS 15-23-01, Underground Radioactive Material Area; and CAS 15-23-03, Contaminated Sump, Piping. All Area 15 CASs are located at the former U.S. Environmental Protection Agency (EPA) Farm, which operated from 1963 to 1981 and was used to support animal experiments involving the uptake of radionuclides. Each of the Area 15 CASs, except CAS 15-23-01, is associated with the disposal of waste effluent from Building 15-06, which was the primary location of the various tests and experiments conducted onsite. Waste effluent disposal from Building 15-06 involved piping, sumps, outfalls, a septic tank with leachfield, underground storage tanks, and an aboveground storage tank (AST). CAS 15-23-01 was associated with decontamination activities of farm equipment potentially contaminated with radiological constituents, pesticides, and herbicides. While the building structures were removed before the investigation took place, all the original tanks, sumps, piping, and concrete building pads remain in place. The Area 6 CAS is located at the Decontamination Facility in Area 6, a facility which operated from 1971 to 2001 and was used to decontaminate vehicles, equipment, clothing, and other materials that had become contaminated during nuclear testing activities. The CAS includes the effluent collection and distribution systems for Buildings

  16. Manipulating the Biosynthesis of Bioactive Compound Alkaloids for Next-Generation Metabolic Engineering in Opium Poppy Using CRISPR-Cas 9 Genome Editing Technology.

    PubMed

    Alagoz, Yagiz; Gurkok, Tugba; Zhang, Baohong; Unver, Turgay

    2016-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) endonuclease system is a powerful RNA-guided genome editing tool. CRISPR/Cas9 has been well studied in model plant species for targeted genome editing. However, few studies have been reported on plant species without whole genome sequence information. Currently, no study has been performed to manipulate metabolic pathways using CRISPR/Cas9. In this study, the type II CRISPR/SpCas9 system was used to knock out, via nonhomologous end-joining genome repair, the 4'OMT2 in opium poppy (Papaver somniferum L.), a gene which regulates the biosythesis of benzylisoquinoline alkaloids (BIAs). For sgRNA transcription, viral-based TRV and synthetic binary plasmids were designed and delivered into plant cells with a Cas9 encoding-synthetic vector by Agrobacterium-mediated transformation. InDels formed by CRISPR/Cas9 were detected by sequence analysis. Our results showed that the biosynthesis of BIAs (e.g. morphine, thebaine) was significantly reduced in the transgenic plants suggesting that 4'OMT2 was efficiently knocked-out by our CRISPR-Cas9 genome editing approach. In addition, a novel uncharacterized alkaloid was observed only in CRISPR/Cas9 edited plants. Thus, the applicabilitiy of the CRISPR/Cas9 system was demonstrated for the first time for medicinal aromatic plants by sgRNAs transcribed from both synthetic and viral vectors to regulate BIA metabolism and biosynthesis. PMID:27483984

  17. A Participatory Action Research Study of Nature Education in Nature: Towards Community-Based Eco-Pedagogy

    ERIC Educational Resources Information Center

    Eryaman, Mustafa Yunus; Yalcin-Ozdilek, Sukran; Okur, Emel; Cetinkaya, Zeynep; Uygun, Selcuk

    2010-01-01

    Contemporary nature education is exploring different ways to develop awareness for change and initiate action. Such educational activities go beyond creating understanding and awareness in order to develop a sense of commitment for individual and collective action. This participatory action research study aimed to improve teachers' sensitiveness…

  18. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  19. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  20. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  1. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  2. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  3. The Environmental Action and Philosophy Matrix: An Exploratory Study of the Environmental Attitudes of Recreation Management and Environmental Studies Students

    ERIC Educational Resources Information Center

    Schultz, Jeremy R.; Simpson, Steven; Elfessi, Abdulaziz M.

    2011-01-01

    This study is a comparative analysis of the environmental philosophies of college undergraduates enrolled in a Midwestern university. Two courses were used for the research, one from a recreation management curriculum and the other from environmental studies. The study utilized a survey instrument called the Environmental Action and Philosophy…

  4. CRISPR-Cas: Revolutionising genome engineering.

    PubMed

    Nicholson, Samantha Anne; Pepper, Michael Sean

    2016-09-01

    The ability to permanently alter or repair the human genome has been the subject of a number of science fiction films, but with the recent advent of several customisable sequence-specific endonuclease technologies, genome engineering looks set to become a clinical reality in the near future. This article discusses recent advancements in the technology called 'clustered regularly interspaced palindromic repeat (CRISPR)-associated genes' (CRISPR-Cas), the potential of CRISPR-Cas to revolutionise molecular medicine, and the ethical and regulatory hurdles facing its application. PMID:27601107

  5. Engineered CRISPR-Cas9 nucleases with altered PAM specificities

    PubMed Central

    Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q.; Topkar, Ved; Nguyen, Nhu T.; Zheng, Zongli; Gonzales, Andrew P.W.; Li, Zhuyun; Peterson, Randall T.; Yeh, Jing-Ruey Joanna; Aryee, Martin J.; Joung, J. Keith

    2015-01-01

    Although CRISPR-Cas9 nucleases are widely used for genome editing1, 2, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM)3–6. As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-Seq analysis7. In addition, we identified and characterized another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also found that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities. PMID:26098369

  6. Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

    PubMed

    Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q; Topkar, Ved V; Nguyen, Nhu T; Zheng, Zongli; Gonzales, Andrew P W; Li, Zhuyun; Peterson, Randall T; Yeh, Jing-Ruey Joanna; Aryee, Martin J; Joung, J Keith

    2015-07-23

    Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities. PMID:26098369

  7. Closure Report for Corrective Action Unit 357: Mud Pits and Waste Dump, Nevada Test Site, Nevada, Rev. No.: 0

    SciTech Connect

    Laura A. Pastor

    2005-04-01

    This Closure Report (CR) presents information supporting closure of Corrective Action Unit (CAU) 357: Mud Pits and Waste Dump, Nevada Test Site (NTS), Nevada. The CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada, U.S. Department of Energy (DOE), and the U.S. Department of Defense (FFACO, 1996). Corrective Action Unit 357 is comprised of 14 Corrective Action Sites (CASs) located in Areas 1, 4, 7, 8, 10, and 25 of the NTS (Figure 1-1). The NTS is located approximately 65 miles (mi) northwest of Las Vegas, Nevada. Corrective Action Unit 357 consists of 11 CASs that are mud pits located in Areas 7, 8, and 10. The mud pits were associated with drilling activities conducted on the NTS in support of the underground nuclear weapons testing. The remaining three CASs are boxes and pipes associated with Building 1-31.2el, lead bricks, and a waste dump. These CAS are located in Areas 1, 4, and 25, respectively. The following CASs are shown on Figure 1-1: CAS 07-09-02, Mud Pit; CAS 07-09-03, Mud Pit; CAS 07-09-04, Mud Pit; CAS 07-09-05, Mud Pit; CAS 08-09-01, Mud Pit; CAS 08-09-02, Mud Pit; CAS 08-09-03, Mud Pit; CAS 10-09-02, Mud Pit; CAS 10-09-04, Mud Pit; CAS 10-09-05, Mud Pit; CAS 10-09-06, Mud Pit, Stains, Material; CAS 01-99-01, Boxes, Pipes; CAS 04-26-03, Lead Bricks; and CAS 25-15-01, Waste Dump. The purpose of the corrective action activities was to obtain analytical data that supports the closure of CAU 357. Environmental samples were collected during the investigation to determine whether contaminants exist and if detected, their extent. The investigation and sampling strategy was designed to target locations and media most likely to be contaminated (biased sampling). A general site conceptual model was developed for each CAS to support and guide the investigation as outlined in the Streamlined Approach for Environmental Restoration (SAFER) Plan (NNSA/NSO, 2003b). This CR

  8. Resolving Leadership Dilemmas in New Zealand Kindergartens: An Action Research Study

    ERIC Educational Resources Information Center

    Cardno, Carol; Reynolds, Bronwyn

    2009-01-01

    Purpose: The purpose of this paper is to examine dilemmas encountered by kindergarten head teachers with the further aim of developing their capability to recognise and resolve "leadership dilemmas". Design/methodology/approach: Action research was used to conduct a three-phase study involving 16 kindergarten head teachers and six system managers…

  9. An Action Research Study of Student Self-Assessment in Higher Education

    ERIC Educational Resources Information Center

    Walser, Tamara M.

    2009-01-01

    Although student self-assessment is considered a critical component of assessment for learning, its use and related research are rare in higher education. This article describes an action research study of self-assessment as an instructional strategy in two university courses. Results indicate that self-assessment exercises provided students the…

  10. How Trainee Music Teachers Learn about Teaching by Talking to Each Other: An Action Research Study

    ERIC Educational Resources Information Center

    Cain, Tim

    2011-01-01

    This article presents an action research study into how trainee music teachers in England use a structured discussion process called "Collegial Consultation" to learn about teaching. The research shows that, in Collegial Consultation, trainees learn from each other by offering several solutions to a problem, offering reasons for their ideas,…

  11. Using Professional Development to Improve Elementary Teachers' Mathematics Teaching: An Action Research Study

    ERIC Educational Resources Information Center

    Good, Leslie

    2009-01-01

    Research indicates that it is common for elementary teachers to possess limited mathematics content knowledge, high levels of math anxiety, and low levels of mathematics teacher efficacy. Assuming action research as the chosen methodology, this research study was developed with the ultimate goal of improving how elementary teachers in my district…

  12. Exploring Student Experiences of Belonging within an Urban High School Choral Ensemble: An Action Research Study

    ERIC Educational Resources Information Center

    Parker, Elizabeth Cassidy

    2010-01-01

    The purpose of this action research study was to describe adolescent singers' experiences of belonging within one urban high school choral ensemble. Understanding student perspectives on belonging within music ensembles can assist choral educators, parents and administrators in order to more fully support adolescent emotional and social…

  13. A Community Organizes for Action: A Case Study of the Mon-Yough Region in Pennsylvania.

    ERIC Educational Resources Information Center

    Avery, Robert W.; Chesler, Herbert A.

    This case study examines the development and problems of the Mon-Yough Community Action Committee, Inc. (MYCAC), one of the local anti-poverty agencies in Allegheny County, Pennsylvania. The agency's major effort is to overcome problems created by the decline of the local steel industry by supporting existing welfare agencies, and through such…

  14. Implementing an Action Research Project: A Case Study in Making Decisions and Managing Challenges

    ERIC Educational Resources Information Center

    Greenbank, Paul

    2013-01-01

    This paper examines the challenges involved in implementing an action research project. It discusses a project which uses a series of interventions (unfreezing techniques, cases studies in conjunction with analogical encoding and lecturer input) to encourage students to critically reflect on their approach to career decision-making. This paper…

  15. A Hermeneutic Phenomenological Study of Community Mural Making and Social Action Art Therapy

    ERIC Educational Resources Information Center

    Rossetto, Erica

    2012-01-01

    Through a hermeneutic phenomenological study of interview data from 8 community artists, the author sought to discover commonalities and differences in the worldviews and philosophies of self that underlie community mural making as they relate to art therapy as social action and art therapy practice within a traditional Western cultural framework.…

  16. Training and Work Organisation: An Action-Research Study in a Sales and Distribution Company

    ERIC Educational Resources Information Center

    Bernardes, Alda Cristina; Lopes, Albino Pedro

    2005-01-01

    This study seeks to define a method of designing work-linked training, based on day-to-day work practices and the collaboration between all those involved. From diagnosis to evaluation, no training is designed or given without considering the opinions and interests of the parties involved. The method used is based on action research (AR) and on…

  17. Developing Pedagogical Content Knowledge of Science Teachers through Action Research: A Case Study from Pakistan

    ERIC Educational Resources Information Center

    Halai, Nelofer; Khan, Manzoor Ali

    2011-01-01

    The aim of this study is to analyze the action research undertaken by Khan as a teacher researcher in a private school as part of the degree requirement of his M.Ed. program in Teacher Education at the Aga Khan University, Institute for Educational Development. The purpose of this analysis undertaken by the first author and supervisor of the study…

  18. An Action Research Study on the Effect of Interactive Technology and Active Learning on Student Performance

    ERIC Educational Resources Information Center

    Bear, Teresa J.

    2013-01-01

    This quantitative action science research study utilized a causal-comparative experimental research design in order to determine if the use of student response systems (clickers), as an active learning strategy in a community college course, improved student performance in the course. Students in the experimental group (n = 26) used clickers to…

  19. Seeking New Ways of Living Community in the Classroom and the World: An Action Research Study

    ERIC Educational Resources Information Center

    Morris, Anne

    2012-01-01

    Action research is a method of examining one's own practice through reflection and critical self-study. In this paper, the author considers her experience teaching a two-day lesson on connections and relationships in William Forsythe's dance "One Flat Thing, reproduced" (2000) and the accompanying website, Synchronous Objects. The author reviews…

  20. A Case Study Examining Change in Teacher Beliefs through Collaborative Action Research

    ERIC Educational Resources Information Center

    Vaino, Katrin; Holbrook, Jack; Rannikmae, Miia

    2013-01-01

    The main goal of this study was to explore the role of collaborative action research in eliciting change in teacher beliefs. The beliefs were those of five chemistry teachers in implementing a new teaching approach, geared to enhancing students' scientific and technological literacy (STL). The teacher beliefs were analysed based on Ajzen's Theory…

  1. Service Learning Experience and Undergraduate Leadership Behaviors: An Action Research Case Study

    ERIC Educational Resources Information Center

    Kenary, Judy

    2010-01-01

    Service-learning has been shown to be an effective practice that positively affects students' academic achievement, self-esteem, and problem-solving skills (Billig, 2002; Eyler & Giles, 1999; Wilczenski & Coomey, 2007). This mixed-method action research case study was conducted to explore the possible link between service-learning experience and…

  2. Learning How to Manage Bias: A Case Study of Youth Participatory Action Research

    ERIC Educational Resources Information Center

    Kirshner, Ben; Pozzoboni, Kristen; Jones, Hannah

    2011-01-01

    Youth programs that are organized around intellectually challenging, socially relevant projects create opportunities for deep cognitive engagement. One type of authentic project that deserves attention from applied developmental scientists is youth participatory action research (YPAR), in which participants study a problem relevant to young…

  3. Effects of Information, Guidance, and Actions on Postsecondary Destinations: A Study of Talent Loss.

    ERIC Educational Resources Information Center

    Plank, Stephen B.; Jordan, Will J.

    2001-01-01

    Uses National Educational Longitudinal Study data for approximately 25,000 students to show that information about higher education, guidance, and essential preparatory actions taken by secondary school students influences whether they will attend postsecondary education institutions within 2 years of high school graduation, and the types of…

  4. Characterizing Teaching Effectiveness in the Joint Action Theory in Didactics: An Exploratory Study in Primary School

    ERIC Educational Resources Information Center

    Sensevy, Gérard

    2014-01-01

    This paper presents an exploratory study of two consecutive reading sessions conducted in primary school by two different teachers. Our purpose is twofold. From a theoretical viewpoint, we propose a tentative set of conditions of teaching effectiveness by relying on the Joint Action Theory in Didactics. From a methodological viewpoint, drawing on…

  5. An Action Research Study of Effective and Efficient Rehearsals in a Grade 8 Band Setting

    ERIC Educational Resources Information Center

    Ferley, Maureen

    2007-01-01

    Involving 28 grade eight students, this action research study examined strategies that lead to effective and efficient band rehearsals at the junior high level. Data was gathered concerning the proportion of instructional time spent on teaching concepts and skills, on active music making, and on classroom management. From this, and a review of the…

  6. Reflective Practice and Motion Sickness: Thoughts on the First North American Action Research Study Day

    ERIC Educational Resources Information Center

    Rowell, Lonnie; Inoue, Noriyuki; Getz, Cheryl

    2014-01-01

    This paper examines the experience of an action research "Study Day" to investigate development of a culture of reflective practice among educators. Shared recognition of the importance of reflective practice in education is now a well-established part of both pre-service preparation and in-service work experience for educators. Osterman…

  7. Exploring a Science Teacher's Uncertainty with Integrating Engineering Design: An Action Research Study

    ERIC Educational Resources Information Center

    Capobianco, Brenda M.

    2011-01-01

    This study examines a fifth grade science teacher's attempts at integrating engineering design using the construct of uncertainty. Collaborative action research served as a supportive mechanism to uncover and confront the teacher's uncertainties. Data were gathered through semi-structured interviews, reflections, classroom observations, lesson…

  8. An Intervention to Increase the Use of Asthma Action Plans in Schools: A MASNRN Study

    ERIC Educational Resources Information Center

    Pulcini, Joyce; DeSisto, Marie C.; McIntyre, C. Lynne

    2007-01-01

    School nurses, in collaboration with primary care providers (PCPs), can work to better manage asthma by using the Asthma Action Plan (AAP) with peak flow monitoring. The aim of this pilot study was to determine the effectiveness of an intervention to increase the number of AAPs in schools for students with asthma by having school nurses provide…

  9. Self-Study in Teaching and Teacher Development: A Call to Action

    ERIC Educational Resources Information Center

    Pithouse, Kathleen; Mitchell, Claudia; Weber, Sandra

    2009-01-01

    This article maps out key features of self-study in teaching and teacher development, particularly in relation to social action. As teacher educator-researchers, we have become increasingly interested in how self-reflexivity in teaching and teacher development can illuminate social and educational challenges that have resonance beyond the self and…

  10. Teachers' Continuation of Action Research Elements after Conducting Studies during a Master's Program

    ERIC Educational Resources Information Center

    Barnes, Johanna S.

    2013-01-01

    Teachers are critical to student learning in the classroom, and just like students, teachers perform better when they are able to make choices based on what is relevant to them. Action research is a way for teachers to systematically inquire and reflect to make necessary improvements in practice for student learning. This study involved teachers…

  11. Interdisciplinary Literacy through Social Media in the Mathematics Classroom: An Action Research Study

    ERIC Educational Resources Information Center

    Casey, Gail

    2013-01-01

    This article looks at how social and participatory media can be used to strengthen interdisciplinary literacy and connects the multimodality of social environments with Middle-Years Mathematics curriculum and delivery. The article reports on part of an eighteen months action research study in an Australian public high school within the…

  12. The Treatment of Maladaptive Shame in Borderline Personality Disorder: A Pilot Study of "Opposite Action"

    ERIC Educational Resources Information Center

    Rizvi, Shireen L.; Linehan, Marsha M.

    2005-01-01

    This study sought to pilot test a short-term intervention for maladaptive shame in borderline personality disorder (BPD) based on the skill of "opposite action" from dialectical behavior therapy. Five women with BPD were treated with the intervention using a single-subject, multiple-baseline design. Results indicate that, although state ratings of…

  13. Habitual vs Non-Habitual Manual Actions: An ERP Study on Overt Movement Execution

    PubMed Central

    Westerholz, Jan; Schack, Thomas; Schütz, Christoph; Koester, Dirk

    2014-01-01

    This study explored the neurophysiological mechanisms underlying the planning and execution of an overt goal-related handle rotation task. More specifically, we studied the neural basis of motor actions concerning the influence of the grasp choice. The aim of the present study was to differentiate cerebral activity between grips executed in a habitual and a non-habitual mode, and between specified and free grip choices. To our knowledge, this is the first study to differentiate cerebral activity underlying overt goal-related actions executed with a focus on the habitual mode. In a handle rotation task, participants had to use thumb-toward (habitual) or thumb-away (non-habitual) grips to rotate a handle to a given target position. Reaction and reach times were shorter for the habitual compared to the non-habitual mode indicating that the habitual mode requires less cognitive processing effort than the non-habitual mode. Neural processes for action execution (measured by event-related potentials (ERPs)) differed between habitual and non-habitual conditions. We found differential activity between habitual and non-habitual conditions in left and right frontal areas from −600 to 200 ms time-locked to reaching the target position. No differential neural activity could be traced for the specification of the grip. The results suggested that the frontal negativity reflected increased difficulty in movement precision control in the non-habitual mode compared to the habitual mode during the homing in phase of grasp and rotation actions. PMID:24691654

  14. Education as Sustainability: An Action Research Study of the Burns Model of Sustainability Pedagogy

    ERIC Educational Resources Information Center

    Burns, Heather

    2009-01-01

    Postsecondary teaching and learning must be reoriented to equip learners with the knowledge, skills, and values they will need for creating a more sustainable world. This action research study examined the effects of implementing the "Burns model of sustainability pedagogy" in university courses taught by the researcher. This model is comprised of…

  15. An Action Research Study Designed to Implement Student Negotiation to Improve Speaking Classroom Practice in Turkey

    ERIC Educational Resources Information Center

    Uztosun, Mehmet Sercan; Skinner, Nigel; Cadorath, Jill

    2014-01-01

    This paper reports the second stage of an action research study designed to improve the effectiveness of speaking classes through negotiating the lesson contents with students. The data were collected through interviews, questionnaires and observations as a way of eliciting students' views. The research, conducted in an English language…

  16. An Action Research Study: Using Classroom Guidance Lessons to Teach Middle School Students about Sexual Harassment

    ERIC Educational Resources Information Center

    Bates, Rebecca C.

    2006-01-01

    This article describes a three-part classroom guidance lesson that teaches middle school students the definition of sexual harassment, the difference between flirting and sexual harassment, and the harmful effects of sexual harassment. An action research study evaluated the effectiveness of the lessons in decreasing referrals for sexual harassment…

  17. Joint Action in Didactics and Classroom Ecology: Comparing Theories Using a Case Study in Physical Education

    ERIC Educational Resources Information Center

    Amade-Escot, Chantal; Venturini, Patrice

    2015-01-01

    This paper examines the respective contribution of two theoretical approaches to teaching and learning: the classroom ecology (CE) framework from Anglo-American research and the joint action in didactics (JAD) framework, which is part of French "didactique" research. This theoretical comparison is grounded in data from a case study in a…

  18. Dialogic Construction and Reflective Practice: A Teacher Educator's Action Research Study of Teacher as Learner.

    ERIC Educational Resources Information Center

    Haley, Marjorie; Wesley-Nero, Sabrina

    This paper reports on an action research study on the use of reflective practice by a teacher educator. It highlights how the teacher educator co-constructed knowledge with a doctoral student within reflective environments, examining teaching practices, and analyzed dialogic perspectives. Using journals as a primary means of reflection allowed the…

  19. An Evaluation of Professional Development to Improve Teachers' Perspectives and Behaviors: An Action Research Study

    ERIC Educational Resources Information Center

    Beckford-Young, Paulette Vivienne

    2016-01-01

    The purpose of this action research study was to conduct a professional development activity to provide content-area teachers with academic vocabulary strategies to be implemented during instruction on a daily basis. Professional development is essential for teachers to gain new knowledge and skills in order to hone their craft to improve student…

  20. Scientist-Teacher Partnerships as Professional Development: An Action Research Study

    ERIC Educational Resources Information Center

    Willcuts, Meredith Harris

    2009-01-01

    The overall purpose of this action research study was to explore the experiences of ten middle school science teachers involved in a three-year partnership program between scientists and teachers at a Department of Energy national laboratory, including the impact of the program on their professional development, and to improve the partnership…

  1. Applying the Participatory Action Research Model to the Study of Social Inclusion at Worksites.

    ERIC Educational Resources Information Center

    Park, Hyun-Sook; Gonsier-Gerdin, Jean; Hoffman, Stacey; Whaley, Susan; Yount, Michael

    1998-01-01

    A study used participatory action research (PAR) to explore social inclusion/relationships at worksites of 10 students (ages 17-21). The participatory intervention process assisted teachers and job coaches in making constructive changes in transition work experience programs to provide social opportunities for students and help them become part of…

  2. We Did It Together: A Participatory Action Research Study on Poverty and Disability

    ERIC Educational Resources Information Center

    Buettgen, Alexis; Richardson, Jason; Beckham, Kristie; Richardson, Kathy; Ward, Michelle; Riemer, Manuel

    2012-01-01

    This article presents the perspective of both non-disabled and developmentally disabled people working together in a research project on poverty and disability. Our study used a participatory action research approach that challenges the norm of exclusion in the research process. Control of the research agenda has been inclusive and shared to…

  3. In-Service Teacher Training and Coaching on Marzano's Instructional Strategies: An Action Research Study

    ERIC Educational Resources Information Center

    Miller, Shenequa C.

    2014-01-01

    The focus of this action research study was the implementation of a professional development initiative comprised of two phases: a training program for teachers on Marzano's nine research-based instructional strategies, and the implementation, supported by follow-up coaching, during "Pear Mountain" High School's (a pseudonym) six-week…

  4. Task-Based Language Learning and Teaching: An Action-Research Study

    ERIC Educational Resources Information Center

    Calvert, Megan; Sheen, Younghee

    2015-01-01

    The creation, implementation, and evaluation of language learning tasks remain a challenge for many teachers, especially those with limited experience with using tasks in their teaching. This action-research study reports on one teacher's experience of developing, implementing, critically reflecting on, and modifying a language learning task…

  5. Improving Middle School Parental Engagement in Transition to Common Core State Standards: An Action Research Study

    ERIC Educational Resources Information Center

    Harla, Donna K.

    2014-01-01

    Parental involvement in schools is an important potential contributor to improving American education and making the U.S. more globally competitive. This qualitative and quantitative mixed-methodology action research study probed the viability of engaging parents around issues of educational improvement by inviting them to participate in training…

  6. Web 2.0 and Language Learners' Motivation: An Action Research Study

    ERIC Educational Resources Information Center

    Anwaruddin, Sardar M.

    2013-01-01

    Based on the observation that most of my students use computer-based technology (CBT) in their daily activities, I used computer assisted language learning (CALL) as an intervention in this action research study, carried out at a university in Bangladesh. This CALL curriculum was focused on Web 2.0 and its applications for educational purposes.…

  7. Investigating the neural basis of cooperative joint action. An EEG hyperscanning study.

    PubMed

    Astolfi, L; Toppi, J; Vogel, P; Mattia, D; Babiloni, F; Ciaramidaro, A; Siniatchkin, M

    2014-01-01

    The aim of the present study is to investigate the neurophysiological basis of the cognitive functions underlying the execution of joint actions, by means of the recent technique called hyperscanning. Neuroelectrical hyperscanning is based on the simultaneous recording of brain activity from multiple subjects and includes the analysis of the functional relation between the brain activity of all the interacting individuals. We recorded simultaneous high density electroencephalography (hdEEG) from 16 pairs of subjects involved in a computerized joint action paradigm, with controlled levels of cooperation. Results of cortical connectivity analysis returned significant differences, in terms of inter-brain functional causal links, between the condition of cooperative joint action and a condition in which the subjects were told they were interacting with a PC, while actually interacting with another human subject. Such differences, described by selected brain connectivity indices, point toward an integration between the two subjects' brain activity in the cooperative condition, with respect to control conditions. PMID:25571089

  8. The association between protective actions and homicide risk: findings from the Oklahoma Lethality Assessment Study.

    PubMed

    Messing, Jill Theresa; Campbell, Jacquelyn C; Brown, Sheryll; Patchell, Beverly; Androff, David K; Wilson, Janet Sullivan

    2014-01-01

    This study focuses on the relationship between women's risk of homicide as measured by the Danger Assessment and 13 protective actions. Participants (N = 432) experienced an incident of police involved intimate partner violence (IPV) and subsequently completed a structured telephone interview. Most women in this sample experienced severe violence and were classified as being at high risk for homicide. Participants engaged in an average of 3.81 (SD = 2.73) protective actions. With the exception of the use of formal domestic violence services, women in the high-risk category were significantly more likely than women in the lower risk category to have used each of the protective actions examined. Implications for research and practice are discussed. PMID:25199385

  9. Regulators of Androgen Action Resource: a one-stop shop for the comprehensive study of androgen receptor action.

    PubMed

    DePriest, Adam D; Fiandalo, Michael V; Schlanger, Simon; Heemers, Frederike; Mohler, James L; Liu, Song; Heemers, Hannelore V

    2016-01-01

    Androgen receptor (AR) is a ligand-activated transcription factor that is the main target for treatment of non-organ-confined prostate cancer (CaP). Failure of life-prolonging AR-targeting androgen deprivation therapy is due to flexibility in steroidogenic pathways that control intracrine androgen levels and variability in the AR transcriptional output. Androgen biosynthesis enzymes, androgen transporters and AR-associated coregulators are attractive novel CaP treatment targets. These proteins, however, are characterized by multiple transcript variants and isoforms, are subject to genomic alterations, and are differentially expressed among CaPs. Determining their therapeutic potential requires evaluation of extensive, diverse datasets that are dispersed over multiple databases, websites and literature reports. Mining and integrating these datasets are cumbersome, time-consuming tasks and provide only snapshots of relevant information. To overcome this impediment to effective, efficient study of AR and potential drug targets, we developed the Regulators of Androgen Action Resource (RAAR), a non-redundant, curated and user-friendly searchable web interface. RAAR centralizes information on gene function, clinical relevance, and resources for 55 genes that encode proteins involved in biosynthesis, metabolism and transport of androgens and for 274 AR-associated coregulator genes. Data in RAAR are organized in two levels: (i) Information pertaining to production of androgens is contained in a 'pre-receptor level' database, and coregulator gene information is provided in a 'post-receptor level' database, and (ii) an 'other resources' database contains links to additional databases that are complementary to and useful to pursue further the information provided in RAAR. For each of its 329 entries, RAAR provides access to more than 20 well-curated publicly available databases, and thus, access to thousands of data points. Hyperlinks provide direct access to gene

  10. Regulators of Androgen Action Resource: a one-stop shop for the comprehensive study of androgen receptor action

    PubMed Central

    Fiandalo, Michael V.; Schlanger, Simon; Heemers, Frederike; Mohler, James L.; Liu, Song; Heemers, Hannelore V.

    2016-01-01

    Androgen receptor (AR) is a ligand-activated transcription factor that is the main target for treatment of non-organ-confined prostate cancer (CaP). Failure of life-prolonging AR-targeting androgen deprivation therapy is due to flexibility in steroidogenic pathways that control intracrine androgen levels and variability in the AR transcriptional output. Androgen biosynthesis enzymes, androgen transporters and AR-associated coregulators are attractive novel CaP treatment targets. These proteins, however, are characterized by multiple transcript variants and isoforms, are subject to genomic alterations, and are differentially expressed among CaPs. Determining their therapeutic potential requires evaluation of extensive, diverse datasets that are dispersed over multiple databases, websites and literature reports. Mining and integrating these datasets are cumbersome, time-consuming tasks and provide only snapshots of relevant information. To overcome this impediment to effective, efficient study of AR and potential drug targets, we developed the Regulators of Androgen Action Resource (RAAR), a non-redundant, curated and user-friendly searchable web interface. RAAR centralizes information on gene function, clinical relevance, and resources for 55 genes that encode proteins involved in biosynthesis, metabolism and transport of androgens and for 274 AR-associated coregulator genes. Data in RAAR are organized in two levels: (i) Information pertaining to production of androgens is contained in a ‘pre-receptor level’ database, and coregulator gene information is provided in a ‘post-receptor level’ database, and (ii) an ‘other resources’ database contains links to additional databases that are complementary to and useful to pursue further the information provided in RAAR. For each of its 329 entries, RAAR provides access to more than 20 well-curated publicly available databases, and thus, access to thousands of data points. Hyperlinks provide direct access to

  11. A case study of the development of environmental action projects from the framework of participatory action research within two middle school classrooms

    NASA Astrophysics Data System (ADS)

    Charmatz, Kim

    The purpose of this study was to understand student and teacher empowerment through a socially critical environmental education perspective. The main research question guiding this study was: How do participants make sense of a learning experience in which students design and carry out an environmental action project in their community? This study used participatory action research and critical theory as practical and theoretical frameworks. These frameworks were relevant as this study sought to examine social change, power, and relationships through participants' experiences. The context of this study was within one seventh and one eighth grade classroom participating in environmental projects. The study was conducted in spring 2005 with an additional follow-up data collection period during spring 2006. The school was located in a densely populated metropolitan suburb. Fifty-three students, a teacher researcher, and three science teachers participated. Data sources were written surveys, scores on Middle School Environmental Literacy Survey Instrument (MSELI), observations, interviews, and student work. This study used a mixed methodological approach. Quantitative data analysis involved dependent samples t-test scores on the MSELI before and after the completion of the projects. Qualitative data were analyzed using an inductive analysis approach. This study has implications for educators interested in democratic education. Environmental action projects provide a context for students and teachers to learn interdisciplinary content knowledge, develop personal beliefs, and learn ways to take action in their communities. This pedagogy has the potential to increase cooperation, communication, and tensions within school communities. Students' participation in the development of environmental action projects may lead to feelings of empowerment or being able to make a difference in their community, as an individual or member of a group. Future research is needed to discern

  12. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2012-10-01 2012-10-01 false CAS applicability....

  13. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2014-10-01 2014-10-01 false Types of CAS coverage....

  14. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false CAS applicability....

  15. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2012-10-01 2012-10-01 false Types of CAS coverage....

  16. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 7 2012-10-01 2012-10-01 false CAS applicability. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  17. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false Types of CAS coverage....

  18. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false CAS applicability. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  19. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false Types of CAS coverage....

  20. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false CAS applicability....

  1. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 7 2014-10-01 2014-10-01 false CAS applicability. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  2. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2014-10-01 2014-10-01 false CAS applicability....

  3. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 7 2013-10-01 2012-10-01 true CAS applicability. 9903.201... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  4. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Types of CAS coverage....

  5. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false CAS applicability....

  6. Corrective Action Investigation Plan for Corrective Action Unit 555: Septic Systems Nevada Test Site, Nevada, Rev. No.: 0 with Errata

    SciTech Connect

    Pastor, Laura

    2005-12-01

    This Corrective Action Investigation Plan (CAIP) contains project-specific information including facility descriptions, environmental sample collection objectives, and criteria for conducting site investigation activities at Corrective Action Unit (CAU) 555: Septic Systems, Nevada Test Site (NTS), Nevada. This CAIP has been developed in accordance with the ''Federal Facility Agreement and Consent Order'' (FFACO) (1996) that was agreed to by the State of Nevada, the U.S. Department of Energy (DOE), and the U.S. Department of Defense. Corrective Action Unit 555 is located in Areas 1, 3 and 6 of the NTS, which is approximately 65 miles (mi) northwest of Las Vegas, Nevada, and is comprised of the five corrective action sites (CASs) shown on Figure 1-1 and listed below: (1) CAS 01-59-01, Area 1 Camp Septic System; (2) CAS 03-59-03, Core Handling Building Septic System; (3) CAS 06-20-05, Birdwell Dry Well; (4) CAS 06-59-01, Birdwell Septic System; and (5) CAS 06-59-02, National Cementers Septic System. An FFACO modification was approved on December 14, 2005, to include CAS 06-20-05, Birdwell Dry Well, as part of the scope of CAU 555. The work scope was expanded in this document to include the investigation of CAS 06-20-05. The Corrective Action Investigation (CAI) will include field inspections, radiological surveys, geophysical surveys, sampling of environmental media, analysis of samples, and assessment of investigation results, where appropriate. Data will be obtained to support corrective action alternative evaluations and waste management decisions. The CASs in CAU 555 are being investigated because hazardous and/or radioactive constituents may be present in concentrations that could potentially pose a threat to human health and the environment. Existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives for the CASs. Additional information will be generated by conducting a CAI

  7. Engineering of Isogenic Cells Deficient for MR1 with a CRISPR/Cas9 Lentiviral System: Tools To Study Microbial Antigen Processing and Presentation to Human MR1-Restricted T Cells.

    PubMed

    Laugel, Bruno; Lloyd, Angharad; Meermeier, Erin W; Crowther, Michael D; Connor, Thomas R; Dolton, Garry; Miles, John J; Burrows, Scott R; Gold, Marielle C; Lewinsohn, David M; Sewell, Andrew K

    2016-08-01

    The nonclassical HLA molecule MHC-related protein 1 (MR1) presents metabolites of the vitamin B synthesis pathways to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells. This new class of Ags represents a variation on the classical paradigm of self/non-self discrimination because these T cells are activated through their TCR by small organic compounds generated during microbial vitamin B2 synthesis. Beyond the fundamental significance, the invariant nature of MR1 across the human population is a tantalizing feature for the potential development of universal immune therapeutic and diagnostic tools. However, many aspects of MR1 Ag presentation and MR1-restricted T cell biology remain unknown, and the ubiquitous expression of MR1 across tissues and cell lines can be a confounding factor for experimental purposes. In this study, we report the development of a novel CRISPR/Cas9 genome editing lentiviral system and its use to efficiently disrupt MR1 expression in A459, THP-1, and K562 cell lines. We generated isogenic MR1(-/-) clonal derivatives of the A549 lung carcinoma and THP-1 monocytic cell lines and used these to study T cell responses to intracellular pathogens. We confirmed that MAIT cell clones were unable to respond to MR1(-/-) clones infected with bacteria whereas Ag presentation by classical and other nonclassical HLAs was unaffected. This system represents a robust and efficient method to disrupt the expression of MR1 and should facilitate investigations into the processing and presentation of MR1 Ags as well as into the biology of MAIT cells. PMID:27307560

  8. Engineering of Isogenic Cells Deficient for MR1 with a CRISPR/Cas9 Lentiviral System: Tools To Study Microbial Antigen Processing and Presentation to Human MR1-Restricted T Cells

    PubMed Central

    Lloyd, Angharad; Meermeier, Erin W.; Crowther, Michael D.; Connor, Thomas R.; Dolton, Garry; Miles, John J.; Burrows, Scott R.; Gold, Marielle C.; Lewinsohn, David M.

    2016-01-01

    The nonclassical HLA molecule MHC-related protein 1 (MR1) presents metabolites of the vitamin B synthesis pathways to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells. This new class of Ags represents a variation on the classical paradigm of self/non-self discrimination because these T cells are activated through their TCR by small organic compounds generated during microbial vitamin B2 synthesis. Beyond the fundamental significance, the invariant nature of MR1 across the human population is a tantalizing feature for the potential development of universal immune therapeutic and diagnostic tools. However, many aspects of MR1 Ag presentation and MR1-restricted T cell biology remain unknown, and the ubiquitous expression of MR1 across tissues and cell lines can be a confounding factor for experimental purposes. In this study, we report the development of a novel CRISPR/Cas9 genome editing lentiviral system and its use to efficiently disrupt MR1 expression in A459, THP-1, and K562 cell lines. We generated isogenic MR1−/− clonal derivatives of the A549 lung carcinoma and THP-1 monocytic cell lines and used these to study T cell responses to intracellular pathogens. We confirmed that MAIT cell clones were unable to respond to MR1−/− clones infected with bacteria whereas Ag presentation by classical and other nonclassical HLAs was unaffected. This system represents a robust and efficient method to disrupt the expression of MR1 and should facilitate investigations into the processing and presentation of MR1 Ags as well as into the biology of MAIT cells. PMID:27307560

  9. Large fragment deletion using a CRISPR/Cas9 system in Saccharomyces cerevisiae.

    PubMed

    Hao, Huanhuan; Wang, Xiaofei; Jia, Haiyan; Yu, Miao; Zhang, Xiaoyu; Tang, Hui; Zhang, Liping

    2016-09-15

    Large chromosomal modifications have been performed in natural and laboratory evolution studies and hold tremendous potential for use in foundational research, medicine, and biotechnology applications. Recently, the type II bacterial Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR-associated (CRISPR/Cas9) system has emerged as a powerful tool for genome editing in various organisms. In this study, we applied the CRISPR/Cas9 system to preform large fragment deletions in Saccharomyces cerevisiae and compared the performance activity to that of a traditional method that uses the Latour system. Here we report in S. Cerevisiae the CRIPR/Cas9 system has been used to delete fragments exceeding 30 kb. The use of the CRISPR/Cas9 system for generating chromosomal segment excision showed some potential advantages over the Latour system. All the results indicated that CRISPR/Cas9 system was a rapid, efficient, low-cost, and versatile method for genome editing and that it can be applied in further studies in the fields of biology, agriculture, and medicine. PMID:27402178

  10. Studies on the mechanisms of action of the herbicide safener CGA-92194

    SciTech Connect

    Zama, P.

    1986-01-01

    CGA-92194 is a herbicide safener that is used as a seed dressing agent to protect grain sorghum against metolachlor injury. The potential adverse phytotoxic effects and the mechanisms of the protective action of this safener were studied in laboratory experiments. Adverse phytotoxicity was assessed by comparing CGA-92194 and the herbicide safeners cyometrinil and flurazole for their effects on CO/sub 2/ fixation, protein, DNA, RNA and lipid synthesis of enzymatically isolated leaf cells of soybean. The safening action mechanisms of CGA-92194 were studied by examining the potential interactions of this safener with metolachlor at the levels of uptake and macromolecular syntheses in enzymatically isolated leaf mesophyll protoplasts of grain sorghum. When CGA-92194 and metolachlor were given simultaneously, CGA-92194 enhanced /sup 14/C-metolachlor uptake into the sorghum protoplasts in a concentration-dependent pattern. Treatments with metolachlor and CGA-92194 in combination inhibited the incorporation of /sup 14/C-uracil, /sup 3/H-thymidine and /sup 14/C-acetate into sorghum protoplast macromolecules less than metolachlor given alone, suggesting the potential involvement of a competitive antagonism in CGA-92194 mechanism of action. The metabolic activity and growth of sorghum seedlings grown from CGA-92194-pretreated seeds were lower than that of seedlings grown from untreated seeds at 10 or 20 days after planting. These results indicate that a safener-induced stimulation of the spontaneous or enzymatic conjugation of metolachlor with GSH is most likely involved in CGA-92194 protective action.

  11. A Case Study Examining Change in Teacher Beliefs Through Collaborative Action Research

    NASA Astrophysics Data System (ADS)

    Vaino, Katrin; Holbrook, Jack; Rannikmäe, Miia

    2013-01-01

    The main goal of this study was to explore the role of collaborative action research in eliciting change in teacher beliefs. The beliefs were those of five chemistry teachers in implementing a new teaching approach, geared to enhancing students' scientific and technological literacy (STL). The teacher beliefs were analysed based on Ajzen's Theory of Planned Behaviour (2005) by looking at the teacher's (a) attitude towards implementing STL modules, (b) perceived subjective norms, and (c) behavioural control regarding the new teaching approach. After an introductory year, when teachers familiarised themselves with the new approach, a collaborative action research project was initiated in the second year of the study, helping teachers to minimise or overcome initially perceived constraints when implementing STL modules in their classroom. The processes of teacher change and the course of the project were investigated by teacher interviews, teacher informal commentaries, and meeting records. The formation of positive beliefs towards a STL approach increased continuously, although its extent and character varied depending on the teacher. The close cooperation, in the format of collaborative action research and especially through teacher group reflections and perceived collegial support, did support teacher professional development including change in their beliefs towards the new teaching approach. Additionally, positive feedback gained from other teachers through running a two-day in-service course in year three helped to strengthen all five teachers' existing beliefs towards the new approach. The current research demonstrated that perceived constraints, where identified, can be meaningfully addressed by teachers, through undertaking collaborative action research.

  12. Generation of porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene by somatic cell nuclear transfer.

    PubMed

    Liu, Guoqian; Liu, Kai; Wei, Hengxi; Li, Li; Zhang, Shouquan

    2016-09-01

    Cas9 endonuclease, from so-called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems of Streptococcus pyogenes, type II functions as an RNA-guided endonuclease and edits the genomes of prokaryotic and eukaryotic organisms, including deletion and insertion by DNA double‑stranded break repair mechanisms. In previous studies, it was observed that Cas9, with a genome‑scale lentiviral single‑guide RNA library, could be applied to a loss‑of‑function genetic screen, although the loss‑of‑function genes have yet to be verified in vitro and this approach has not been used in porcine cells. Based on these observations, lentiviral Cas9 was used to infect porcine primary fibroblasts to achieve cell colonies carrying Cas9 endonuclease. Subsequently, porcine fetal fibroblasts expressing the tetracycline‑inducible Cas9 gene were generated by somatic cell nuclear transfer, and three 30 day transgenic porcine fetal fibroblasts (PFFs) were obtained. Polymerase chain reaction (PCR), reverse transcription‑PCR and western blot analysis indicated that the PFFs were Cas9‑positive. In addition, one of the three integrations was located near to known functional genes in the PFF1 cell line, whereas neither of the integrations was located in the PFF1 or PFF2 cell lines. It was hypothesized that these transgenic PFFs may be useful for conditional genomic editing in pigs, and for generating ideal modified porcine models. PMID:27430306

  13. CRISPR/Cas9 produces anti-hepatitis B virus effect in hepatoma cells and transgenic mouse.

    PubMed

    Zhu, Wei; Xie, Kun; Xu, Yuanjian; Wang, Le; Chen, Kaiming; Zhang, Longzhen; Fang, Jianmin

    2016-06-01

    Chronic infection of hepatitis B virus (HBV) is at risk of liver cirrhosis and hepatocellular carcinoma and remains one of the major public health problems worldwide. It is a major barrier of persistence HBV cccDNA under current antiviral therapy as novel strategies of disrupting HBV cccDNA is pressing. The (CRISPR)/Cas9 system is presently emerging in gene editing and we also apply it for targeting and deleting the conserved regions of HBV genome. Two homologous sequences of HBV S and X genes were carried with CRISPR/Cas9 endonuclease to build pCas9 constructs, which may mediate anti-HBV effects of in vitro and in vivo systems in this study. The results showed the better anti-HBV productions by pCas9-2 and without significant differences in between Huh7 and HepG2 cells. CRISPR/Cas9 direct cleavage and mutagenesis were further analyzed of in vitro system. In the M-TgHBV mouse model of HBV, injection of pCas9 constructs by hydrodynamics decreased HBsAg of sera and liver HBcAg. In conclusion, this designed CRISPR/Cas9 system can induce anti-HBV effects and potentially consider as a novel therapeutic agent against chronic HBV infection. PMID:27049051

  14. Generation of porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene by somatic cell nuclear transfer

    PubMed Central

    Liu, Guoqian; Liu, Kai; Wei, Hengxi; Li, Li; Zhang, Shouquan

    2016-01-01

    Cas9 endonuclease, from so-called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems of Streptococcus pyogenes, type II functions as an RNA-guided endonuclease and edits the genomes of prokaryotic and eukaryotic organisms, including deletion and insertion by DNA double-stranded break repair mechanisms. In previous studies, it was observed that Cas9, with a genome-scale lentiviral single-guide RNA library, could be applied to a loss-of-function genetic screen, although the loss-of-function genes have yet to be verified in vitro and this approach has not been used in porcine cells. Based on these observations, lentiviral Cas9 was used to infect porcine primary fibroblasts to achieve cell colonies carrying Cas9 endonuclease. Subsequently, porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene were generated by somatic cell nuclear transfer, and three 30 day transgenic porcine fetal fibroblasts (PFFs) were obtained. Polymerase chain reaction (PCR), reverse transcription-PCR and western blot analysis indicated that the PFFs were Cas9-positive. In addition, one of the three integrations was located near to known functional genes in the PFF1 cell line, whereas neither of the integrations was located in the PFF1 or PFF2 cell lines. It was hypothesized that these transgenic PFFs may be useful for conditional genomic editing in pigs, and for generating ideal modified porcine models. PMID:27430306

  15. Cas-enabled technologies as `agents provocateurs' in teaching and learning mathematical modelling in secondary school classrooms

    NASA Astrophysics Data System (ADS)

    Geiger, Vince; Faragher, Rhonda; Goos, Merrilyn

    2010-09-01

    This paper draws on a one year study of three secondary school classrooms to examine the nature of student-student-technology interaction when working in partnership with computer algebra systems (CAS) on mathematical modelling tasks and the classroom affordances and constraints that influence such interaction. The analysis of these data indicates that CAS enabled technologies have a role to play as provocateurs of productive student-student-teacher interaction in both small group and whole class settings. Our research indicates that technologies that incorporate CAS capabilities have the potential to mediate collaborative approaches to mathematical enquiry within life-related mathematical tasks.

  16. CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA.

    PubMed

    Zhou, Jianting; Wu, Ronghai; Xue, Xiaoli; Qin, Zhongjun

    2016-08-19

    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

  17. Closure Report for Corrective Action Unit 543: Liquid Disposal Units, Nevada Test Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2008-01-01

    This Closure Report (CR) documents closure activities for Corrective Action Unit (CAU) 543, Liquid Disposal Units, according to the Federal Facility Agreement and Consent Order (FFACO, 1996) and the Corrective Action Plan (CAP) for CAU 543 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2007). CAU 543 is located at the Nevada Test Site (NTS), Nevada (Figure 1), and consists of the following seven Corrective Action Sites (CASs): CAS 06-07-01, Decon Pad; CAS 15-01-03, Aboveground Storage Tank; CAS 15-04-01, Septic Tank; CAS 15-05-01, Leachfield; CAS 15-08-01, Liquid Manure Tank; CAS 15-23-01, Underground Radioactive Material Area; CAS 15-23-03, Contaminated Sump, Piping; and CAS 06-07-01 is located at the Decontamination Facility in Area 6, adjacent to Yucca Lake. The remaining CASs are located at the former U.S. Environmental Protection Agency (EPA) Farm in Area 15. The purpose of this CR is to provide a summary of the completed closure activities, to document waste disposal, and to present analytical data confirming that the remediation goals were met. The closure alternatives consisted of closure in place for two of the CASs, and no further action with implementation of best management practices (BMPs) for the remaining five CASs.

  18. L'enseignement du francais commercial au niveau elementaire: utilisation du "cas professionnel" (Elementary Level Commercial French Language Instruction: Use of the Professional Case Study).

    ERIC Educational Resources Information Center

    Dany, Max; Noe, Christine

    1985-01-01

    Methodological, pedagogical, and psychological reasons for using professional case studies as a teaching technique in elementary business French are discussed, and specific strategies for teaching specific language functions (referential, phatic, metalinguistic, and expressive) are described. (MSE)

  19. Sensitivity of Alpha and Beta Oscillations to Sensorimotor Characteristics of Action: An EEG Study of Action Production and Gesture Observation

    PubMed Central

    Quandt, Lorna C.; Marshall, Peter J.; Shipley, Thomas F.; Beilock, Sian L.; Goldin-Meadow, Susan

    2012-01-01

    The sensorimotor experiences we gain when performing an action have been found to influence how our own motor systems are activated when we observe others performing that same action. Here we asked whether this phenomenon applies to the observation of gesture. Would the sensorimotor experiences we gain when performing an action on an object influence activation in our own motor systems when we observe others performing a gesture for that object? Participants were given sensorimotor experience with objects that varied in weight, and then observed video clips of an actor producing gestures for those objects. Electroencephalography (EEG) was recorded while participants first observed either an iconic gesture (pantomiming lifting an object) or a deictic gesture (pointing to an object) for an object, and then grasped and lifted the object indicated by the gesture. We analyzed EEG during gesture observation to determine whether oscillatory activity was affected by the observer’s sensorimotor experiences with the object represented in the gesture. Seeing a gesture for an object previously experienced as light was associated with a suppression of power in alpha and beta frequency bands, particularly at posterior electrodes. A similar pattern was found when participants lifted the light object, but over more diffuse electrodes. Moreover, alpha and beta bands at right parieto-occipital electrodes were sensitive to the type of gesture observed (iconic vs. deictic). These results demonstrate that sensorimotor experience with an object affects how a gesture for that object is processed, as measured by the gesture-observer’s EEG, and suggest that different types of gestures recruit the observer’s own motor system in different ways. PMID:22910276

  20. Closure Report for Corrective Action Unit 563: Septic Systems, Nevada Test Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2010-02-28

    Corrective Action Unit (CAU) 563 is identified in the Federal Facility Agreement and Consent Order (FFACO) as “Septic Systems” and consists of the following four Corrective Action Sites (CASs), located in Areas 3 and 12 of the Nevada Test Site: · CAS 03-04-02, Area 3 Subdock Septic Tank · CAS 03-59-05, Area 3 Subdock Cesspool · CAS 12-59-01, Drilling/Welding Shop Septic Tanks · CAS 12-60-01, Drilling/Welding Shop Outfalls Closure activities were conducted from September to November 2009 in accordance with the FFACO (1996, as amended February 2008) and the Corrective Action Plan for CAU 563. The corrective action alternatives included No Further Action and Clean Closure.

  1. 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

  2. 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

  3. Structure and Engineering of Francisella novicida Cas9.

    PubMed

    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

    2016-02-25

    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 the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox. PMID:26875867

  4. Tandem repeat knockout utilizing the CRISPR/Cas9 system in human cells.

    PubMed

    Lv, Qingyan; Lai, Liangxue; Yuan, Lin; Song, Yuning; Sui, Tingting; Li, Zhanjun

    2016-05-15

    Tandem repeats have been shown to cause human genetic diseases and contribute significantly to genome variation and instability. Although multi-sgRNAs mediated CRISPR/Cas9 system have used to generate regional deletions previously, in this study we explored a method of generating regional deletions of tandem repeats by taking advantage of the off-target effects of CRISPR/Cas9 in 293FT cells. Our results revealed that generation of large-fragment deletions of tandem repeats located in the MAGEL2 and XIST gene was possible. In summary, we have demonstrated that large-fragment deletions of tandem repeats can be achieved using a sgRNA-directed CRISPR/Cas9 system, facilitating the functional study of tandem repeats in future studies. PMID:26873114

  5. Can discrete joint action be synergistic? Studying the stabilization of interpersonal hand coordination.

    PubMed

    Romero, Veronica; Kallen, Rachel; Riley, Michael A; Richardson, Michael J

    2015-10-01

    The human perceptual-motor system is tightly coupled to the physical and informational dynamics of a task environment. These dynamics operate to constrain the high-dimensional order of the human movement system into low-dimensional, task-specific synergies-functional groupings of structural elements that are temporarily constrained to act as a single coordinated unit. The aim of the current study was to determine whether synergistic processes operate when coacting individuals coordinate to perform a discrete joint-action task. Pairs of participants sat next to each other and each used 1 arm to complete a pointer-to-target task. Using the uncontrolled manifold (UCM) analysis for the first time in a discrete joint action, the structure of joint-angle variance was examined to determine whether there was synergistic organization of the degrees of freedom employed at the interpersonal or intrapersonal levels. The results revealed that the motor actions performed by coactors were synergistically organized at both the interpersonal and intrapersonal levels. More importantly, however, the interpersonal synergy was found to be significantly stronger than the intrapersonal synergies. Accordingly, the results provide clear evidence that coacting individuals can become temporarily organized to form single synergistic 2-person systems during performance of a discrete joint action. PMID:26052696

  6. Can Discrete Joint Action Be Synergistic? Studying the Stabilization of Interpersonal Hand Coordination

    PubMed Central

    Romero, Veronica; Kallen, Rachel; Riley, Michael A.; Richardson, Michael J.

    2016-01-01

    The human perceptual-motor system is tightly coupled to the physical and informational dynamics of a task environment. These dynamics operate to constrain the high-dimensional order of the human movement system into low-dimensional, task-specific synergies—functional groupings of structural elements that are temporarily constrained to act as a single coordinated unit. The aim of the current study was to determine whether synergistic processes operate when coacting individuals coordinate to perform a discrete joint-action task. Pairs of participants sat next to each other and each used 1 arm to complete a pointer-to-target task. Using the uncontrolled manifold (UCM) analysis for the first time in a discrete joint action, the structure of joint-angle variance was examined to determine whether there was synergistic organization of the degrees of freedom employed at the interpersonal or intrapersonal levels. The results revealed that the motor actions performed by coactors were synergistically organized at both the interpersonal and intrapersonal levels. More importantly, however, the interpersonal synergy was found to be significantly stronger than the intrapersonal synergies. Accordingly, the results provide clear evidence that coacting individuals can become temporarily organized to form single synergistic 2-person systems during performance of a discrete joint action. PMID:26052696

  7. Photoactivatable CRISPR-Cas9 for optogenetic genome editing.

    PubMed

    Nihongaki, Yuta; Kawano, Fuun; Nakajima, Takahiro; Sato, Moritoshi

    2015-07-01

    We describe an engineered photoactivatable Cas9 (paCas9) that enables optogenetic control of CRISPR-Cas9 genome editing in human cells. paCas9 consists of split Cas9 fragments and photoinducible dimerization domains named Magnets. In response to blue light irradiation, paCas9 expressed in human embryonic kidney 293T cells induces targeted genome sequence modifications through both nonhomologous end joining and homology-directed repair pathways. Genome editing activity can be switched off simply by extinguishing the light. We also demonstrate activation of paCas9 in spatial patterns determined by the sites of irradiation. Optogenetic control of targeted genome editing should facilitate improved understanding of complex gene networks and could prove useful in biomedical applications. PMID:26076431

  8. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems

    PubMed Central

    2011-01-01

    Background The CRISPR-Cas adaptive immunity systems that are present in most Archaea and many Bacteria function by incorporating fragments of alien genomes into specific genomic loci, transcribing the inserts and using the transcripts as guide RNAs to destroy the genome of the cognate virus or plasmid. This RNA interference-like immune response is mediated by numerous, diverse and rapidly evolving Cas (CRISPR-associated) proteins, several of which form the Cascade complex involved in the processing of CRISPR transcripts and cleavage of the target DNA. Comparative analysis of the Cas protein sequences and structures led to the classification of the CRISPR-Cas systems into three Types (I, II and III). Results A detailed comparison of the available sequences and structures of Cas proteins revealed several unnoticed homologous relationships. The Repeat-Associated Mysterious Proteins (RAMPs) containing a distinct form of the RNA Recognition Motif (RRM) domain, which are major components of the CRISPR-Cas systems, were classified into three large groups, Cas5, Cas6 and Cas7. Each of these groups includes many previously uncharacterized proteins now shown to adopt the RAMP structure. Evidence is presented that large subunits contained in most of the CRISPR-Cas systems could be homologous to Cas10 proteins which contain a polymerase-like Palm domain and are predicted to be enzymatically active in Type III CRISPR-Cas systems but inactivated in Type I systems. These findings, the fact that the CRISPR polymerases, RAMPs and Cas2 all contain core RRM domains, and distinct gene arrangements in the three types of CRISPR-Cas systems together provide for a simple scenario for origin and evolution of the CRISPR-Cas machinery. Under this scenario, the CRISPR-Cas system originated in thermophilic Archaea and subsequently spread horizontally among prokaryotes. Conclusions Because of the extreme diversity of CRISPR-Cas systems, in-depth sequence and structure comparison continue to

  9. An EEG study on the somatotopic organisation of sensorimotor cortex activation during action execution and observation in infancy

    PubMed Central

    de Klerk, Carina C.J.M.; Johnson, Mark H.; Southgate, Victoria

    2015-01-01

    Previous studies have shown that sensorimotor cortex activation is somatotopically-organised during action execution and observation in adulthood. Here we aimed to investigate the development of this phenomenon in infancy. We elicited arm and leg actions from 12-month-old infants and presented them, and a control group of adults, with videos of arm and leg actions while we measured their sensorimotor alpha suppression using EEG. Sensorimotor alpha suppression during action execution was somatotopically organised in 12-month-old infants: there was more suppression over the arm areas when infants performed reaching actions, and more suppression over the leg area when they performed kicking actions. Adults also showed somatotopically-organised activation during the observation of reaching and kicking actions. In contrast, infants did not show somatotopically-organised activation during action observation, but instead activated the arm areas when observing both reaching and kicking actions. We suggest that the somatotopic organisation of sensorimotor cortex activation during action observation may depend on infants’ understanding of the action goal and their expectations about how this goal will be achieved. PMID:26318840

  10. Pseudorabies virus can escape from CRISPR-Cas9-mediated inhibition.

    PubMed

    Peng, Zhiyuan; Ouyang, Ting; Pang, Daxin; Ma, Teng; Chen, Xinrong; Guo, Ning; Chen, Fuwang; Yuan, Lin; Ouyang, Hongsheng; Ren, Linzhu

    2016-09-01

    The CRISPR-Cas9 system is a newly developed genome-engineering tool used to inhibit virus infection by targeting the conserved regions of the viral genomic DNA. In the present study, we constructed a cell line stably expressing Cas9 endonuclease and sgRNA targeting the conserved UL30 gene of pseudorabies virus (PRV). During the PRV infection, the CRISPR-Cas9 system was efficient in cleaving the UL30 gene in each passage. However, deletions and insertions occurred at low passages, while substitutions were frequently observed at high passages. Furthermore, copy numbers and virus titers of PRV were significantly increased in a passage-dependent manner, indicating that viral genomic replication and assembly were more effective at the high passages than at low passages. These results demonstrated that PRV could escape from CRISPR-Cas9-mediated inhibition. Therefore, whether the CRISPR-Cas9 system is suitable for antiviral application should be considered and carefully verified. PMID:27507009

  11. Friendly Fire: Biological Functions and Consequences of Chromosomal Targeting by CRISPR-Cas Systems.

    PubMed

    Heussler, Gary E; O'Toole, George A

    2016-05-15

    Clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems in bacteria and archaea target foreign elements, such as bacteriophages and conjugative plasmids, through the incorporation of short sequences (termed spacers) from the foreign element into the CRISPR array, thereby allowing sequence-specific targeting of the invader. Thus, CRISPR-Cas systems are typically considered a microbial adaptive immune system. While many of these incorporated spacers match targets on bacteriophages and plasmids, a noticeable number are derived from chromosomal DNA. While usually lethal to the self-targeting bacteria, in certain circumstances, these self-targeting spacers can have profound effects in regard to microbial biology, including functions beyond adaptive immunity. In this minireview, we discuss recent studies that focus on the functions and consequences of CRISPR-Cas self-targeting, including reshaping of the host population, group behavior modification, and the potential applications of CRISPR-Cas self-targeting as a tool in microbial biotechnology. Understanding the effects of CRISPR-Cas self-targeting is vital to fully understanding the spectrum of function of these systems. PMID:26929301

  12. Overcoming doxorubicin resistance of cancer cells by Cas9-mediated gene disruption

    PubMed Central

    Ha, Jong Seong; Byun, Juyoung; Ahn, Dae-Ro

    2016-01-01

    In this study, Cas9 system was employed to down-regulate mdr1 gene for overcoming multidrug resistance of cancer cells. Disruption of the MDR1 gene was achieved by delivery of the Cas9-sgRNA plasmid or the Cas9-sgRNA ribonucleoprotein complex using a conventional gene transfection agent and protein transduction domain (PTD). Doxorubicin showed considerable cytotoxicity to the drug-resistant breast cancer cells pre-treated with the RNA-guided endonuclease (RGEN) systems, whereas virtually non-toxic to the untreated cells. The potency of drug was enhanced in the cells treated with the protein-RNA complex as well as in those treated with plasmids, suggesting that mutation of the mdr1 gene by intracellular delivery of Cas9-sgRNA complex using proper protein delivery platforms could recover the drug susceptibility. Therefore, Cas9-mediated disruption of the drug resistance-related gene can be considered as a promising way to overcome multidrug resistance in cancer cells. PMID:26961701

  13. Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

    PubMed Central

    Ethier, Sylvain; Schmeing, T. Martin; Dostie, Josée; Pelletier, Jerry

    2014-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5′NGG3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data. PMID:25275497

  14. Versatile in vivo regulation of tumor phenotypes by dCas9-mediated transcriptional perturbation.

    PubMed

    Braun, Christian J; Bruno, Peter M; Horlbeck, Max A; Gilbert, Luke A; Weissman, Jonathan S; Hemann, Michael T

    2016-07-01

    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

  15. Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system

    PubMed Central

    Liu, Rui; Chen, Ling; Jiang, Yanping; Zhou, Zhihua; Zou, Gen

    2015-01-01

    Filamentous fungi have wide applications in biotechnology. The CRISPR/Cas9 system is a powerful genome-editing method that facilitates genetic alterations of genomes in a variety of organisms. However, a genome-editing approach has not been reported in filamentous fungi. Here, we demonstrated the establishment of a CRISPR/Cas9 system in the filamentous fungus Trichoderma reesei by specific codon optimization and in vitro RNA transcription. It was shown that the CRISPR/Cas9 system was controllable and conditional through inducible Cas9 expression. This system generated site-specific mutations in target genes through efficient homologous recombination, even using short homology arms. This system also provided an applicable and promising approach to targeting multiple genes simultaneously. Our results illustrate that the CRISPR/Cas9 system is a powerful genome-manipulating tool for T. reesei and most likely for other filamentous fungal species, which may accelerate studies on functional genomics and strain improvement in these filamentous fungi.

  16. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system.

    PubMed

    Xie, Kabin; Minkenberg, Bastian; Yang, Yinong

    2015-03-17

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA-gRNA architecture were efficiently and precisely processed into gRNAs with desired 5' targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits. PMID:25733849

  17. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

    PubMed Central

    Xie, Kabin; Minkenberg, Bastian

    2015-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits. PMID:25733849

  18. Observation and imitation of actions performed by humans, androids, and robots: an EMG study

    PubMed Central

    Hofree, Galit; Urgen, Burcu A.; Winkielman, Piotr; Saygin, Ayse P.

    2015-01-01

    Understanding others’ actions is essential for functioning in the physical and social world. In the past two decades research has shown that action perception involves the motor system, supporting theories that we understand others’ behavior via embodied motor simulation. Recently, empirical approach to action perception has been facilitated by using well-controlled artificial stimuli, such as robots. One broad question this approach can address is what aspects of similarity between the observer and the observed agent facilitate motor simulation. Since humans have evolved among other humans and animals, using artificial stimuli such as robots allows us to probe whether our social perceptual systems are specifically tuned to process other biological entities. In this study, we used humanoid robots with different degrees of human-likeness in appearance and motion along with electromyography (EMG) to measure muscle activity in participants’ arms while they either observed or imitated videos of three agents produce actions with their right arm. The agents were a Human (biological appearance and motion), a Robot (mechanical appearance and motion), and an Android (biological appearance and mechanical motion). Right arm muscle activity increased when participants imitated all agents. Increased muscle activation was found also in the stationary arm both during imitation and observation. Furthermore, muscle activity was sensitive to motion dynamics: activity was significantly stronger for imitation of the human than both mechanical agents. There was also a relationship between the dynamics of the muscle activity and motion dynamics in stimuli. Overall our data indicate that motor simulation is not limited to observation and imitation of agents with a biological appearance, but is also found for robots. However we also found sensitivity to human motion in the EMG responses. Combining data from multiple methods allows us to obtain a more complete picture of action

  19. Observation and imitation of actions performed by humans, androids, and robots: an EMG study.

    PubMed

    Hofree, Galit; Urgen, Burcu A; Winkielman, Piotr; Saygin, Ayse P

    2015-01-01

    Understanding others' actions is essential for functioning in the physical and social world. In the past two decades research has shown that action perception involves the motor system, supporting theories that we understand others' behavior via embodied motor simulation. Recently, empirical approach to action perception has been facilitated by using well-controlled artificial stimuli, such as robots. One broad question this approach can address is what aspects of similarity between the observer and the observed agent facilitate motor simulation. Since humans have evolved among other humans and animals, using artificial stimuli such as robots allows us to probe whether our social perceptual systems are specifically tuned to process other biological entities. In this study, we used humanoid robots with different degrees of human-likeness in appearance and motion along with electromyography (EMG) to measure muscle activity in participants' arms while they either observed or imitated videos of three agents produce actions with their right arm. The agents were a Human (biological appearance and motion), a Robot (mechanical appearance and motion), and an Android (biological appearance and mechanical motion). Right arm muscle activity increased when participants imitated all agents. Increased muscle activation was found also in the stationary arm both during imitation and observation. Furthermore, muscle activity was sensitive to motion dynamics: activity was significantly stronger for imitation of the human than both mechanical agents. There was also a relationship between the dynamics of the muscle activity and motion dynamics in stimuli. Overall our data indicate that motor simulation is not limited to observation and imitation of agents with a biological appearance, but is also found for robots. However we also found sensitivity to human motion in the EMG responses. Combining data from multiple methods allows us to obtain a more complete picture of action

  20. Differential Distribution of Type II CRISPR-Cas Systems in Agricultural and Nonagricultural Campylobacter coli and Campylobacter jejuni Isolates Correlates with Lack of Shared Environments

    PubMed Central

    Pearson, Bruce M.; Louwen, Rogier; van Baarlen, Peter; van Vliet, Arnoud H.M.

    2015-01-01

    CRISPR (clustered regularly interspaced palindromic repeats)-Cas (CRISPR-associated) systems are sequence-specific adaptive defenses against phages and plasmids which are widespread in prokaryotes. Here we have studied whether phylogenetic relatedness or sharing of environmental niches affects the distribution and dissemination of Type II CRISPR-Cas systems, first in 132 bacterial genomes from 15 phylogenetic classes, ranging from Proteobacteria to Actinobacteria. There was clustering of distinct Type II CRISPR-Cas systems in phylogenetically distinct genera with varying G+C%, which share environmental niches. The distribution of CRISPR-Cas within a genus was studied using a large collection of genome sequences of the closely related Campylobacter species Campylobacter jejuni (N = 3,746) and Campylobacter coli (N = 486). The Cas gene cas9 and CRISPR-repeat are almost universally present in C. jejuni genomes (98.0% positive) but relatively rare in C. coli genomes (9.6% positive). Campylobacter jejuni and agricultural C. coli isolates share the C. jejuni CRISPR-Cas system, which is closely related to, but distinct from the C. coli CRISPR-Cas system found in C. coli isolates from nonagricultural sources. Analysis of the genomic position of CRISPR-Cas insertion suggests that the C. jejuni-type CRISPR-Cas has been transferred to agricultural C. coli. Conversely, the absence of the C. coli-type CRISPR-Cas in agricultural C. coli isolates may be due to these isolates not sharing the same environmental niche, and may be affected by farm hygiene and biosecurity practices in the agricultural sector. Finally, many CRISPR spacer alleles were linked with specific multilocus sequence types, suggesting that these can assist molecular epidemiology applications for C. jejuni and C. coli. PMID:26338188

  1. Differential Distribution of Type II CRISPR-Cas Systems in Agricultural and Nonagricultural Campylobacter coli and Campylobacter jejuni Isolates Correlates with Lack of Shared Environments.

    PubMed

    Pearson, Bruce M; Louwen, Rogier; van Baarlen, Peter; van Vliet, Arnoud H M

    2015-09-01

    CRISPR (clustered regularly interspaced palindromic repeats)-Cas (CRISPR-associated) systems are sequence-specific adaptive defenses against phages and plasmids which are widespread in prokaryotes. Here we have studied whether phylogenetic relatedness or sharing of environmental niches affects the distribution and dissemination of Type II CRISPR-Cas systems, first in 132 bacterial genomes from 15 phylogenetic classes, ranging from Proteobacteria to Actinobacteria. There was clustering of distinct Type II CRISPR-Cas systems in phylogenetically distinct genera with varying G+C%, which share environmental niches. The distribution of CRISPR-Cas within a genus was studied using a large collection of genome sequences of the closely related Campylobacter species Campylobacter jejuni (N = 3,746) and Campylobacter coli (N = 486). The Cas gene cas9 and CRISPR-repeat are almost universally present in C. jejuni genomes (98.0% positive) but relatively rare in C. coli genomes (9.6% positive). Campylobacter jejuni and agricultural C. coli isolates share the C. jejuni CRISPR-Cas system, which is closely related to, but distinct from the C. coli CRISPR-Cas system found in C. coli isolates from nonagricultural sources. Analysis of the genomic position of CRISPR-Cas insertion suggests that the C. jejuni-type CRISPR-Cas has been transferred to agricultural C. coli. Conversely, the absence of the C. coli-type CRISPR-Cas in agricultural C. coli isolates may be due to these isolates not sharing the same environmental niche, and may be affected by farm hygiene and biosecurity practices in the agricultural sector. Finally, many CRISPR spacer alleles were linked with specific multilocus sequence types, suggesting that these can assist molecular epidemiology applications for C. jejuni and C. coli. PMID:26338188

  2. Examination of mechanisms underlying enhanced memory performance in action video game players: a pilot study

    PubMed Central

    Li, Xianchun; Cheng, Xiaojun; Li, Jiaying; Pan, Yafeng; Hu, Yi; Ku, Yixuan

    2015-01-01

    Previous studies have shown enhanced memory performance resulting from extensive action video game playing. The mechanisms underlying the cognitive benefit were investigated in the current study. We presented two types of retro-cues, with variable intervals to memory array (Task 1) or test array (Task 2), during the retention interval in a change detection task. In Task 1, action video game players demonstrated steady performance while non-action video game players showed decreased performance as cues occurred later, indicating their performance difference increased as the cue-to-memory-array intervals became longer. In Task 2, both participant groups increased their performance at similar rates as cues presented later, implying the performance difference in two groups were irrespective of the test-array-to-cue intervals. These findings suggested that memory benefit from game plays is not attributable to the higher ability of overcoming interference from the test array, but to the interactions between the two processes of protection from decay and resistance from interference, or from alternative hypotheses. Implications for future studies were discussed. PMID:26136720

  3. Examination of mechanisms underlying enhanced memory performance in action video game players: a pilot study.

    PubMed

    Li, Xianchun; Cheng, Xiaojun; Li, Jiaying; Pan, Yafeng; Hu, Yi; Ku, Yixuan

    2015-01-01

    Previous studies have shown enhanced memory performance resulting from extensive action video game playing. The mechanisms underlying the cognitive benefit were investigated in the current study. We presented two types of retro-cues, with variable intervals to memory array (Task 1) or test array (Task 2), during the retention interval in a change detection task. In Task 1, action video game players demonstrated steady performance while non-action video game players showed decreased performance as cues occurred later, indicating their performance difference increased as the cue-to-memory-array intervals became longer. In Task 2, both participant groups increased their performance at similar rates as cues presented later, implying the performance difference in two groups were irrespective of the test-array-to-cue intervals. These findings suggested that memory benefit from game plays is not attributable to the higher ability of overcoming interference from the test array, but to the interactions between the two processes of protection from decay and resistance from interference, or from alternative hypotheses. Implications for future studies were discussed. PMID:26136720

  4. CRISPR/Cas9 system as an innovative genetic engineering tool: Enhancements in sequence specificity and delivery methods.

    PubMed

    Jo, Young-Il; Suresh, Bharathi; Kim, Hyongbum; Ramakrishna, Suresh

    2015-12-01

    While human gene therapy has gained significant attention for its therapeutic promise, CRISPR/Cas9 technology has made a breakthrough as an efficient genome editing tool by emulating prokaryotic immune defense mechanisms. Although many studies have found that CRISPR/Cas9 technology is more efficient, specific and manipulable than previous generations of gene editing tools, it can be further improved by elevating its overall efficiency in a higher frequency of genome modifications and reducing its off-target effects. Here, we review the development of CRISPR/Cas9 technology, focusing on enhancement of its sequence specificity, reduction of off-target effects and delivery systems. Moreover, we describe recent successful applications of CRISPR/Cas9 technology in laboratory and clinical studies. PMID:26434948

  5. Closure Report for Corrective Action Unit 104: Area 7 Yucca Flat Atmospheric Test Sites, Nevada National Security Site, Nevada

    SciTech Connect

    2013-06-27

    This Closure Report (CR) presents information supporting closure of Corrective Action Unit (CAU) 104, Area 7 Yucca Flat Atmospheric Test Sites, and provides documentation supporting the completed corrective actions and confirmation that closure objectives for CAU 104 were met. This CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; the U.S. Department of Energy (DOE), Environmental Management; the U.S. Department of Defense; and DOE, Legacy Management. CAU 104 consists of the following 15 Corrective Action Sites (CASs), located in Area 7 of the Nevada National Security Site: · CAS 07-23-03, Atmospheric Test Site T-7C · CAS 07-23-04, Atmospheric Test Site T7-1 · CAS 07-23-05, Atmospheric Test Site · CAS 07-23-06, Atmospheric Test Site T7-5a · CAS 07-23-07, Atmospheric Test Site - Dog (T-S) · CAS 07-23-08, Atmospheric Test Site - Baker (T-S) · CAS 07-23-09, Atmospheric Test Site - Charlie (T-S) · CAS 07-23-10, Atmospheric Test Site - Dixie · CAS 07-23-11, Atmospheric Test Site - Dixie · CAS 07-23-12, Atmospheric Test Site - Charlie (Bus) · CAS 07-23-13, Atmospheric Test Site - Baker (Buster) · CAS 07-23-14, Atmospheric Test Site - Ruth · CAS 07-23-15, Atmospheric Test Site T7-4 · CAS 07-23-16, Atmospheric Test Site B7-b · CAS 07-23-17, Atmospheric Test Site - Climax Closure activities began in October 2012 and were completed in April 2013. Activities were conducted according to the Corrective Action Decision Document/Corrective Action Plan for CAU 104. The corrective actions included No Further Action and Clean Closure. Closure activities generated sanitary waste, mixed waste, and recyclable material. Some wastes exceeded land disposal limits and required treatment prior to disposal. Other wastes met land disposal restrictions and were disposed in appropriate onsite landfills. The U.S. Department of Energy, National Nuclear Security Administration Nevada Field Office

  6. CRISPR/Cas9 for plant genome editing: accomplishments, problems and prospects.

    PubMed

    Paul, Joseph W; Qi, Yiping

    2016-07-01

    The increasing burden of the world population on agriculture requires the development of more robust crops. Dissecting the basic biology that underlies plant development and stress responses will inform the design of better crops. One powerful tool for studying plants at the molecular level is the RNA-programmed genome editing system composed of a clustered regularly interspaced short palindromic repeats (CRISPR)-encoded guide RNA and the nuclease Cas9. Here, some of the recent advances in CRISPR/Cas9 technology that have profound implications for improving the study of plant biology are described. These tools are also paving the way towards new horizons for biotechnologies and crop development. PMID:27114166

  7. The CARN/ARNA Inaugural Study Day Inquiry: What Happens to Action Research after the Master's Degree?

    ERIC Educational Resources Information Center

    Shosh, Joseph M.; McAteer, Mary

    2016-01-01

    The Collaborative Action Research Network (CARN) held its first American study day on the east coast of the United States in conjunction with the Action Research Network of the Americas (ARNA) 2014 conference in Bethlehem, Pennsylvania, USA. Study day participants visited three American secondary schools, one each in Pennsylvania, New York, and…

  8. Participatory Action Research and the Reconstruction of Teachers' Practical Thinking: Lesson Studies and Core Reflection. An Experience in Spain

    ERIC Educational Resources Information Center

    Perez, Angel I.; Soto, Encarnacion; Servan, M. Jose

    2010-01-01

    Following the thoughts and topics we have discussed and worked on for a very long time with Bridget Somekh, we would like to present the theoretical relationship between lesson studies, action research and practical knowledge in teacher education. Inspired by the pedagogical philosophy of lesson studies, participatory action research, and core…

  9. Carcinogenesis Bioassay of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (CAS No. 1746-01-6) in Swiss-Webster Mice (Dermal Study).

    PubMed

    1982-02-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin occurs as a highly toxic impurity found in herbicides containing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4,5-T- derivatives, as well as in other chemicals synthesized using 2,4,5-trichlorophenol. The herbicide 2,4,5-T has been marketed in the United States since 1948. Production increased sharply between 1960 and 1970 when a 1:1 mixture of 2,4,5-T and 2,4-dichlorophenoxyacetic acid (2,4-D) was used as a defoliant in Vietnam under the names of "herbicide agent orange, herbicide orange, agent orange, and orange". During this 10-year period, about 106 million pounds of 2,4,5-T were sprayed. A carcinogenesis bioassay was conducted by applying an acetone suspension of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the clipped backs of 30 male and female Swiss-Webster mice 3 days per week for 99 or 104 weeks. Similar groups were pretreated with 1 application of 50 &mgr;g dimethylbenzanthracene (DMBA) in 0.1 ml acetone 1 week before TCDD administration began. Female mice received 0.005 &mgr;g TCDD per application, and the male mice received 0.001 &mgr;g TCDD. As vehicle controls, 45 mice of each sex received 0.1 ml acetone three times per week. Thirty animals of each sex were used as untreated controls. Throughout the bioassay, mean body weights of the male and female mice administered TCDD, or TCDD following DMBA, were essentially the same as those of the corresponding vehicle control group. Mean body weights of dosed and vehicle control groups of males were less thanthose of the untreated control group throughout the study; for the females, mean body weights were less than the untreated controls during the first 80 weeks. In female mice, the incidences of fibrosarcoma in the integumentary system in dosed groups with TCDD were significantly (P=0.007) higher than that in the corresponding controls (2/41, 5%; 8/27, 30%). An increase in the same tumor type, although not statistically significant (P=0.084), was also observed in

  10. Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing in rats by inhibiting NHEJ and using Cas9 protein.

    PubMed

    Ma, Yuanwu; Chen, Wei; Zhang, Xu; Yu, Lei; Dong, Wei; Pan, Shuo; Gao, Shan; Huang, Xingxu; Zhang, Lianfeng

    2016-07-01

    Precise modifications such as site mutation, codon replacement, insertion or precise targeted deletion are needed for studies of accurate gene function. The CRISPR/Cas9 system has been proved as a powerful tool to generate gene knockout and knockin animals. But the homologous recombination (HR)-directed precise genetic modification mediated by CRISPR/Cas9 is relatively lower compared with nonhomologous end-joining (NHEJ) pathway and extremely expected to be improved. Here, in this study 2 strategies were used to increase the precise genetic modification in rats. Scr7, a DNA ligase IV inhibitor, first identified as an anti-cancer compound, and considered as a potential NHEJ inhibitor, was used to increase the HR-mediated precise genetic modification. Meanwhile, the Cas9 protein instead of mRNA was used to save the mRNA to protein translation step to improve the precise modification efficiency. The Fabp2 and Dbndd1 loci were selected to knockin Cre and CreER(T2), respectively. Our result showed that both Scr7 and Cas9 protein can increase the precise modification. PMID:27163284

  11. Closure Report for Corrective Action Unit 139: Waste Disposal Sites, Nevada Test Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2009-07-31

    Corrective Action Unit (CAU) 139 is identified in the Federal Facility Agreement and Consent Order (FFACO) as 'Waste Disposal Sites' and consists of the following seven Corrective Action Sites (CASs), located in Areas 3, 4, 6, and 9 of the Nevada Test Site: CAS 03-35-01, Burn Pit; CAS 04-08-02, Waste Disposal Site; CAS 04-99-01, Contaminated Surface Debris; CAS 06-19-02, Waste Disposal Site/Burn Pit; CAS 06-19-03, Waste Disposal Trenches; CAS 09-23-01, Area 9 Gravel Gertie; and CAS 09-34-01, Underground Detection Station. Closure activities were conducted from December 2008 to April 2009 according to the FFACO (1996, as amended February 2008) and the Corrective Action Plan for CAU 139 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office, 2007b). The corrective action alternatives included No Further Action, Clean Closure, and Closure in Place with Administrative Controls. Closure activities are summarized. CAU 139, 'Waste Disposal Sites,' consists of seven CASs in Areas 3, 4, 6, and 9 of the NTS. The closure alternatives included No Further Action, Clean Closure, and Closure in Place with Administrative Controls. This CR provides a summary of completed closure activities, documentation of waste disposal, and confirmation that remediation goals were met. The following site closure activities were performed at CAU 139 as documented in this CR: (1) At CAS 03-35-01, Burn Pit, soil and debris were removed and disposed as LLW, and debris was removed and disposed as sanitary waste. (2) At CAS 04-08-02, Waste Disposal Site, an administrative UR was implemented. No postings or post-closure monitoring are required. (3) At CAS 04-99-01, Contaminated Surface Debris, soil and debris were removed and disposed as LLW, and debris was removed and disposed as sanitary waste. (4) At CAS 06-19-02, Waste Disposal Site/Burn Pit, no work was performed. (5) At CAS 06-19-03, Waste Disposal Trenches, a native soil cover was installed, and a UR was

  12. NTP Carcinogenesis Bioassay of Diallyl Phthalate (CAS No. 131-17-9) in B6C3F1 Mice (Gavage Study).

    PubMed

    1983-04-01

    , hyperplasia, and inflammatory lesions of the forestomach were observed in diallyl phthalate-dosed mice of both sexes in a dose-related manner. Papillomas of the forestomach were observed in 0%, 2%, and 4% of the control, low-dose, and high-dose mice of both sexes. The recent historical incidence of this tumor in gavage control mice from both the performing laboratory and other laboratories within the Bioassay Program was less than 1%. Forestomach hyperplasia was diagnosed in 0%, 15%, and 18%, and in 8%, 2%, and 29% of the control, low-dose, and high-dose male and female mice, respectively; chronic inflammation of the forestomach was diagnosed in 0%, 9%, and 16% and in 4%, 2%, and 18% of the control, low-dose, and high-dose male and female mice, respectively. Because of the numerical elevation of the forestomach papillomas in the high-dose mice of both sexes, the concomitant observation of dose related forestomach hyperplasia, and the rarity of this tumor in corn oil (gavage) control B6C3F1 mice, the development of squamous cell papillomas of the forestomach may have been related to diallyl phthalate administration. Under the conditions of this bioassay, the development of chronic inflammation and hyperplasia of the forestomach in both male and female B6C3F1 mice was considered to be related to the administration of diallyl phthalate. The development of squamous cell papillomas of the forestomach may also have been related to chemical administration, but the available data are insufficient to indicate a clear cause and effect relationship. An increase in the incidence of male mice with lymphomas was observed, but this increase was considered only to be equivocally related to diallyl phthalate administration. The results of this bioassay, therefore, do not indicate that diallyl phthalate is carcinogenic in B6C3F1 mice, although a maximum tolerated dose may not have been achieved. A carcinogenicity study by the National Toxicology Program of diallyl phthalate in male and female

  13. 78 FR 58382 - Notice of Final Federal Agency Actions on the Proposed U.S. 50 Study Crossing Over Sinepauxent...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-23

    ...This notice announces actions taken by the FHWA and other Federal agencies that are final within the meaning of 23 U.S.C. 139(l)(1). The action relates to the U.S. 50 Crossing over Sinepauxent Bay Study from MD 611 to MD 378 and 5th Street to Somerset Street located in the Town of Ocean City, Worcester County, Maryland. This action grants approval for the...

  14. ParticipACTION: Baseline assessment of the capacity available to the 'New ParticipACTION': A qualitative study of Canadian organizations

    PubMed Central

    2009-01-01

    Background Evaluation of the original ParticipACTION campaign effects focused on individual awareness, recall, and understanding. Less studied has been the impact such campaigns have had on the broader organizational capacity to mobilize and advocate for physical activity. With the relaunch of ParticipACTION, the purpose of this study was to qualitatively explore baseline organizational capacity to promote physical activity messages, programs, and services within the Canadian context. Methods Using a purposeful sampling strategy, we conducted semi-structured telephone interviews with 49 key informants representing a range of national, provincial, and local organizations with a mandate to promote physical activity. Interview data were analysed using a thematic analytic approach. Results Key informants painted a generally positive picture of current organizational capacity to promote physical activity messages, programs, and services in Canada. Will and leadership were clear strengths while infrastructure limitations remained the greatest concern. Some specific challenges included: 1) funding issues: the absence of core funding in a climate of shifting funding priorities; 2) the difficulty of working without a national physical activity policy (lack of leadership); 3) inconsistent provincial and educational sector level policies; and 4) a persistent focus on obesity rather than physical inactivity. Conclusion The data generated here can be utilized to monitor the future impact of ParticipACTION on enhancing and utilizing this organizational capacity. A range of indicators are suggested that could be used to illustrate ParticipACTION's impact on the broad field of physical activity promotion in the future. PMID:19995458

  15. Visual exploration patterns of human figures in action: an eye tracker study with art paintings

    PubMed Central

    Villani, Daniela; Morganti, Francesca; Cipresso, Pietro; Ruggi, Simona; Riva, Giuseppe; Gilli, Gabriella

    2015-01-01

    Art exploration is a complex process conditioned by factors at different levels and includes both basic visual principles and complex cognitive factors. The human figure is considered a critical factor attracting the attention in art painting. Using an eye-tracking methodology, the goal of this study was to explore different elements of the human figure performing an action (face and body parts in action) in complex social scenes characterized by different levels of social interaction between agents depicted in scenes (individual vs. social). The sample included 44 laypersons, and the stimuli consisted of 10 fine art paintings representing the figurative style of classical art. The results revealed different scanning patterns of the human figure elements related to the level of social interaction of agents depicted in the scene. The agents’ face attracted eye movements in social interaction scenes while the agents’ body parts attracted eye movements only when the agents were involved in individual actions. These processes were confirmed specifically in participants with high empathic abilities who became immediately fixated on faces to develop a mimetic engagement with other agents. Future studies integrating other measures would help confirm the results obtained and strengthen their implication for embodiment processes. PMID:26579021

  16. CERCLA interim action at the Par Pond unit: A case study

    SciTech Connect

    Hickey, H.M.; Matthews, S.S.; Neal, L.W.; Weiss, W.R.

    1993-11-01

    The Par Pond unit designated under CERCLA consists of sediments within a Savannah River Site (SRS) cooling water reservoir. The sediments are contaminated with radionuclides and nonradioactive constituents from nuclear production reactor operations. The mercury in Par Pond is believed to have originated from the Savannah River. Because of Par Pond Dam safety Issues, the water level of the reservoir was drawn down, exposing more than 1300 acres of contaminated sediments and triggering the need for CERCLA interim remedial action. This paper presents the interim action approach taken with Par Pond as a case study. The approach considered the complexity of the Par Pond ecosystem, the large size of Par Pond, the volume of contaminated sediments, and the institutional controls existing at SRS. The Environmental Protection Agency (EPA) considers units with large volumes of low-concentration wastes, as is the case with Par Pond, to be {open_quotes}special sites.{close_quotes} Accordingly, EPA guidance establishes that the range of alternatives developed focus primarily on containment options and other remedial approaches that mitigate potential risks associated with the {open_quotes}special site.{close_quotes} The remedial alternatives, according to EPA, are not to be prohibitively expensive or difficult to implement. This case study also is representative of the types of issues that will need to be addressed within the Department of Energy (DOE) complex as nuclear facilities are transitioned to inactive status and corrective/remedial actions are warranted.

  17. Corrective Action Plan for Corrective Action Unit 262: Area 25 Septic Systems and Underground Discharge Point, Nevada Test Site, Nevada

    SciTech Connect

    K. B. Campbell

    2002-06-01

    This Corrective Action Plan (CAP) provides selected corrective action alternatives and proposes the closure methodology for Corrective Action Unit (CAU) 262, Area 25 Septic Systems and Underground Discharge Point. CAU 262 is identified in the Federal Facility Agreement and Consent Order (FFACO) of 1996. Remediation of CAU 262 is required under the FFACO. CAU 262 is located in Area 25 of the Nevada Test Site (NTS), approximately 100 kilometers (km) (62 miles [mi]) northwest of Las Vegas, Nevada. The nine Corrective Action Sites (CASs) within CAU 262 are located in the Nuclear Rocket Development Station complex. Individual CASs are located in the vicinity of the Reactor Maintenance, Assembly, and Disassembly (R-MAD); Engine Maintenance, Assembly, and Disassembly (E-MAD); and Test Cell C compounds. CAU 262 includes the following CASs as provided in the FFACO (1996); CAS 25-02-06, Underground Storage Tank; CAS 25-04-06, Septic Systems A and B; CAS 25-04-07, Septic System; CAS 25-05-03, Leachfield; CAS 25-05-05, Leachfield; CAS 25-05-06, Leachfield; CAS 25-05-08, Radioactive Leachfield; CAS 25-05-12, Leachfield; and CAS 25-51-01, Dry Well. Figures 2, 3, and 4 show the locations of the R-MAD, the E-MAD, and the Test Cell C CASs, respectively. The facilities within CAU 262 supported nuclear rocket reactor engine testing. Activities associated with the program were performed between 1958 and 1973. However, several other projects used the facilities after 1973. A significant quantity of radioactive and sanitary waste was produced during routine operations. Most of the radioactive waste was managed by disposal in the posted leachfields. Sanitary wastes were disposed in sanitary leachfields. Septic tanks, present at sanitary leachfields (i.e., CAS 25-02-06,2504-06 [Septic Systems A and B], 25-04-07, 25-05-05,25-05-12) allowed solids to settle out of suspension prior to entering the leachfield. Posted leachfields do not contain septic tanks. All CASs located in CAU 262 are

  18. Studies on the Mode of Action of Acifluorfen-Methyl in Nonchlorophyllous Soybean Cells 1

    PubMed Central

    Matringe, M.; Scalla, R.

    1988-01-01

    Phytotoxic effects of the herbicide acifluorfen-methyl on nonchlorophyllous soybean cells were estimated by 86Rb leakage. An action spectrum study showed maximum injury at 350 to 450 nanometers, with lesser activity between 450 and 700 nanometers. Cells treated in the dark with acifluorfen-methyl accumulated fluorescent pigments with the spectral characteristics of protoporphyrin IX. The action spectrum of acifluorfen-methyl matched the absorption spectrum of this tetrapyrrole, and the extent of cellular damage in the light was related to the degree of fluorescent pigment accumulation. We propose that the phytotoxicity of diphenyl ether herbicides could be explained by their ability to cause abnormal accumulations of tetrapyrroles, which in turn induce lethal photooxidative reactions. PMID:16665956

  19. [Studying of sclerosing action of the foam form of polidokanol in experiment in vivo].

    PubMed

    Palamarchuk, V I; Bondarenko, Iu I; Titova, Iu P; Irkin, I V; Zarits'ka, V I

    2014-08-01

    The results of investigation on impact of the foam form of sclerosant in experiment on 80 mongrel white rats, were presented. Sclerosing action of 0.5%, 2%, 3% solution of polydocanol in a microfoam form was studied after its infusion into lateral caudal vein of the rat. Quantity of preparation 0,5 cm3 was determined in accordance to the vein volume. Clinical and macroscopic estimation of the preparation action on internal organs and caudal rat's vein was conducted on the 10th min, every day during 1 week and on the 30th day. When the foam form of sclerosant is introduced intravenously there was not observed thrombotic complications of posterior vena cava and specific macroscopic changes in internal organs, not depending from its concentration, what witnesses security of the method, and selection of the sclerosant solution concentration must depend on the vessel diameter. PMID:25417296

  20. Protein engineering of Cas9 for enhanced function

    PubMed Central

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

    2015-01-01

    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 complimentary 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 directly into 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. PMID:25398355

  1. CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri.

    PubMed

    Oh, Jee-Hwan; van Pijkeren, Jan-Peter

    2014-01-01

    Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR-Cas systems, such as the Streptococcus pyogenes CRISPR-Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR-Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR-Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR-Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR-Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR-Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR-Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria. PMID:25074379

  2. Expanding the catalog of cas genes with metagenomes.

    PubMed

    Zhang, Quan; Doak, Thomas G; Ye, Yuzhen

    2014-02-01

    The CRISPR (clusters of regularly interspaced short palindromic repeats)-Cas adaptive immune system is an important defense system in bacteria, providing targeted defense against invasions of foreign nucleic acids. CRISPR-Cas systems consist of CRISPR loci and cas (CRISPR-associated) genes: sequence segments of invaders are incorporated into host genomes at CRISPR loci to generate specificity, while adjacent cas genes encode proteins that mediate the defense process. We pursued an integrated approach to identifying putative cas genes from genomes and metagenomes, combining similarity searches with genomic neighborhood analysis. Application of our approach to bacterial genomes and human microbiome datasets allowed us to significantly expand the collection of cas genes: the sequence space of the Cas9 family, the key player in the recently engineered RNA-guided platforms for genome editing in eukaryotes, is expanded by at least two-fold with metagenomic datasets. We found genes in cas loci encoding other functions, for example, toxins and antitoxins, confirming the recently discovered potential of coupling between adaptive immunity and the dormancy/suicide systems. We further identified 24 novel Cas families; one novel family contains 20 proteins, all identified from the human microbiome datasets, illustrating the importance of metagenomics projects in expanding the diversity of cas genes. PMID:24319142

  3. 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

  4. Major bacterial lineages are essentially devoid of CRISPR-Cas viral defence systems.

    PubMed

    Burstein, David; Sun, Christine L; Brown, Christopher T; Sharon, Itai; Anantharaman, Karthik; Probst, Alexander J; Thomas, Brian C; Banfield, Jillian F

    2016-01-01

    Current understanding of microorganism-virus interactions, which shape the evolution and functioning of Earth's ecosystems, is based primarily on cultivated organisms. Here we investigate thousands of viral and microbial genomes recovered using a cultivation-independent approach to study the frequency, variety and taxonomic distribution of viral defence mechanisms. CRISPR-Cas systems that confer microorganisms with immunity to viruses are present in only 10% of 1,724 sampled microorganisms, compared with previous reports of 40% occurrence in bacteria and 81% in archaea. We attribute this large difference to the lack of CRISPR-Cas systems across major bacterial lineages that have no cultivated representatives. We correlate absence of CRISPR-Cas with lack of nucleotide biosynthesis capacity and a symbiotic lifestyle. Restriction systems are well represented in these lineages and might provide both non-specific viral defence and access to nucleotides. PMID:26837824

  5. 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

  6. CRISPR/Cas9-Mediated Genome Editing of Mouse Small Intestinal Organoids.

    PubMed

    Schwank, Gerald; Clevers, Hans

    2016-01-01

    The CRISPR/Cas9 system is an RNA-guided genome-editing tool that has been recently developed based on the bacterial CRISPR-Cas immune defense system. Due to its versatility and simplicity, it rapidly became the method of choice for genome editing in various biological systems, including mammalian cells. Here we describe a protocol for CRISPR/Cas9-mediated genome editing in murine small intestinal organoids, a culture system in which somatic stem cells are maintained by self-renewal, while giving rise to all major cell types of the intestinal epithelium. This protocol allows the study of gene function in intestinal epithelial homeostasis and pathophysiology and can be extended to epithelial organoids derived from other internal mouse and human organs. PMID:27246017

  7. Transgene-free genome editing by germline injection of CRISPR/Cas RNA.

    PubMed

    Schwartz, Hillel T; Sternberg, Paul W

    2014-01-01

    Genome modification by CRISPR/Cas offers its users the ability to target endogenous sites in the genome for cleavage and for engineering precise genomic changes using template-directed repair, all with unprecedented ease and flexibility of targeting. As such, CRISPR/Cas is just part of a set of recently developed and rapidly improving tools that offer great potential for researchers to functionally access the genomes of organisms that have not previously been extensively used in a laboratory setting. We describe in detail protocols for using CRISPR/Cas to target genes of experimental organisms, in a manner that does not require transformation to obtain transgenic lines and that should be readily applicable to a wide range of previously little-studied species. PMID:25398352

  8. Major bacterial lineages are essentially devoid of CRISPR-Cas viral defence systems

    PubMed Central

    Burstein, David; Sun, Christine L.; Brown, Christopher T.; Sharon, Itai; Anantharaman, Karthik; Probst, Alexander J.; Thomas, Brian C.; Banfield, Jillian F.

    2016-01-01

    Current understanding of microorganism–virus interactions, which shape the evolution and functioning of Earth's ecosystems, is based primarily on cultivated organisms. Here we investigate thousands of viral and microbial genomes recovered using a cultivation-independent approach to study the frequency, variety and taxonomic distribution of viral defence mechanisms. CRISPR-Cas systems that confer microorganisms with immunity to viruses are present in only 10% of 1,724 sampled microorganisms, compared with previous reports of 40% occurrence in bacteria and 81% in archaea. We attribute this large difference to the lack of CRISPR-Cas systems across major bacterial lineages that have no cultivated representatives. We correlate absence of CRISPR-Cas with lack of nucleotide biosynthesis capacity and a symbiotic lifestyle. Restriction systems are well represented in these lineages and might provide both non-specific viral defence and access to nucleotides. PMID:26837824

  9. Delivery and Specificity of CRISPR-Cas9 Genome Editing Technologies for Human Gene Therapy.

    PubMed

    Gori, Jennifer L; Hsu, Patrick D; Maeder, Morgan L; Shen, Shen; Welstead, G Grant; Bumcrot, David

    2015-07-01

    Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) technology is revolutionizing the study of gene function and likely will give rise to an entire new class of therapeutics for a wide range of diseases. Achieving this goal requires not only characterization of the technology for efficacy and specificity but also optimization of its delivery to the target cells for each disease indication. In this review we survey the various methods by which the CRISPR-Cas9 components have been delivered to cells and highlight some of the more clinically relevant approaches. Additionally, we discuss the methods available for assessing the specificity of Cas9 editing; an important safety consideration for development of the technology. PMID:26068008

  10. T cell-specific inactivation of mouse CD2 by CRISPR/Cas9

    PubMed Central

    Beil-Wagner, Jane; Dössinger, Georg; Schober, Kilian; vom Berg, Johannes; Tresch, Achim; Grandl, Martina; Palle, Pushpalatha; Mair, Florian; Gerhard, Markus; Becher, Burkhard; Busch, Dirk H.; Buch, Thorsten

    2016-01-01

    The CRISPR/Cas9 system can be used to mutate target sequences by introduction of double-strand breaks followed by imprecise repair. To test its use for conditional gene editing we generated mice transgenic for CD4 promoter-driven Cas9 combined with guide RNA targeting CD2. We found that within CD4+ and CD8+ lymphocytes from lymph nodes and spleen 1% and 0.6% were not expressing CD2, respectively. T cells lacking CD2 carryied mutations, which confirmed that Cas9 driven by cell-type specific promoters can edit genes in the mouse and may thus allow targeted studies of gene function in vivo. PMID:26903281

  11. T cell-specific inactivation of mouse CD2 by CRISPR/Cas9.

    PubMed

    Beil-Wagner, Jane; Dössinger, Georg; Schober, Kilian; vom Berg, Johannes; Tresch, Achim; Grandl, Martina; Palle, Pushpalatha; Mair, Florian; Gerhard, Markus; Becher, Burkhard; Busch, Dirk H; Buch, Thorsten

    2016-01-01

    The CRISPR/Cas9 system can be used to mutate target sequences by introduction of double-strand breaks followed by imprecise repair. To test its use for conditional gene editing we generated mice transgenic for CD4 promoter-driven Cas9 combined with guide RNA targeting CD2. We found that within CD4(+) and CD8(+) lymphocytes from lymph nodes and spleen 1% and 0.6% were not expressing CD2, respectively. T cells lacking CD2 carryied mutations, which confirmed that Cas9 driven by cell-type specific promoters can edit genes in the mouse and may thus allow targeted studies of gene function in vivo. PMID:26903281

  12. Major bacterial lineages are essentially devoid of CRISPR-Cas viral defence systems

    DOE PAGESBeta

    Burstein, David; Sun, Christine L.; Brown, Christopher T.; Sharon, Itai; Anantharaman, Karthik; Probst, Alexander J.; Thomas, Brian C.; Banfield, Jillian F.

    2016-02-03

    Here, current understanding of microorganism–virus interactions, which shape the evolution and functioning of Earth’s ecosystems, is based primarily on cultivated organisms. Here we investigate thousands of viral and microbial genomes recovered using a cultivation independent approach to study the frequency, variety and taxonomic distribution of viral defence mechanisms. CRISPR-Cas systems that confer microorganisms with immunity to viruses are present in only 10% of 1,724 sampled microorganisms, compared with previous reports of 40% occurrence in bacteria and 81% in archaea. We attribute this large difference to the lack of CRISPR-Cas systems across major bacterial lineages that have no cultivated representatives. Wemore » correlate absence of CRISPR-Cas with lack of nucleotide biosynthesis capacity and a symbiotic lifestyle. Restriction systems are well represented in these lineages and might provide both non-specific viral defence and access to nucleotides.« less

  13. Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice

    PubMed Central

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2016-01-01

    Recent reports of CRISPR- (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) mediated heritable mutagenesis in plants highlight the need for accuracy of the mutagenesis directed by this system. Off-target mutations are an important issue when considering functional gene analysis, as well as the molecular breeding of crop plants with large genome size, i.e. with many duplicated genes, and where the whole-genome sequence is still lacking. In mammals, off-target mutations can be suppressed by using Cas9 paired nickases together with paired guide RNAs (gRNAs). However, the performance of Cas9 paired nickases has not yet been fully assessed in plants. Here, we analyzed on- and off-target mutation frequency in rice calli and regenerated plants using Cas9 nuclease or Cas9 nickase with paired gRNAs. When Cas9 paired nickases were used, off-target mutations were fully suppressed in rice calli and regenerated plants. However, on-target mutation frequency also decreased compared with that induced by the Cas9 paired nucleases system. Since the gRNA sequence determines specific binding of Cas9 protein–gRNA ribonucleoproteins at the targeted sequence, the on-target mutation frequency of Cas9 paired nickases depends on the design of paired gRNAs. Our results suggest that a combination of gRNAs that can induce mutations at high efficiency with Cas9 nuclease should be used together with Cas9 nickase. Furthermore, we confirmed that a combination of gRNAs containing a one nucleotide (1 nt) mismatch toward the target sequence could not induce mutations when expressed with Cas9 nickase. Our results clearly show the effectiveness of Cas9 paired nickases in delivering on-target specific mutations. PMID:26936792

  14. Breaking-Cas-interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes.

    PubMed

    Oliveros, Juan C; Franch, Mònica; Tabas-Madrid, Daniel; San-León, David; Montoliu, Lluis; Cubas, Pilar; Pazos, Florencio

    2016-07-01

    The CRISPR/Cas technology is enabling targeted genome editing in multiple organisms with unprecedented accuracy and specificity by using RNA-guided nucleases. A critical point when planning a CRISPR/Cas experiment is the design of the guide RNA (gRNA), which directs the nuclease and associated machinery to the desired genomic location. This gRNA has to fulfil the requirements of the nuclease and lack homology with other genome sites that could lead to off-target effects. Here we introduce the Breaking-Cas system for the design of gRNAs for CRISPR/Cas experiments, including those based in the Cas9 nuclease as well as others recently introduced. The server has unique features not available in other tools, including the possibility of using all eukaryotic genomes available in ENSEMBL (currently around 700), placing variable PAM sequences at 5' or 3' and setting the guide RNA length and the scores per nucleotides. It can be freely accessed at: http://bioinfogp.cnb.csic.es/tools/breakingcas, and the code is available upon request. PMID:27166368

  15. CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C

    PubMed Central

    Tsukamoto, Kentaro; Ozeki, Chikako; Kohda, Tomoko; Tsuji, Takao

    2015-01-01

    Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9) system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins. PMID:26177297

  16. Action observers implicitly expect actors to act goal-coherently, even if they do not: an fMRI study.

    PubMed

    Hrkać, Mari; Wurm, Moritz F; Schubotz, Ricarda I

    2014-05-01

    Actions observed in everyday life normally consist of one person performing sequences of goal-directed actions. The present fMRI study tested the hypotheses that observers are influenced by the actor's identity, even when this information is task-irrelevant, and that this information shapes their expectation on subsequent actions of the same actor. Participants watched short video clips of action steps that either pertained to a common action with an overarching goal or not, and were performed by either one or by varying actors (2 × 2 design). Independent of goal coherence, actor coherence elicited activation in dorsolateral and ventromedial frontal cortex, together pointing to a spontaneous attempt to integrate all actions performed by one actor. Interestingly, watching an actor performing unrelated actions elicited additional activation in left inferior frontal gyrus, suggesting a search in semantic memory in an attempt to construct an overarching goal that can reconcile the disparate action steps with a coherent intention. Post-experimental surveys indicate that these processes occur mostly unconsciously. Findings strongly suggest a spontaneous expectation bias toward actor-related episodes in action observers, and hence to the immense impact of actor information on action observation. PMID:23983202

  17. Entrepreneurial Learning through Action: A Case Study of the Six-Squared Program

    ERIC Educational Resources Information Center

    Pittaway, Luke; Missing, Caroline; Hudson, Nigel; Maragh, Dean

    2009-01-01

    This paper explores the role of "action" in entrepreneurial learning and illustrates how programs designed to support action learning can enhance management development in entrepreneurial businesses. The paper begins by exploring action learning and the way "action" is conceived in different types of program. In the second part, the paper details…

  18. Conditionally Stabilized dCas9 Activator for Controlling Gene Expression in Human Cell Reprogramming and Differentiation

    PubMed Central

    Balboa, Diego; Weltner, Jere; Eurola, Solja; Trokovic, Ras; Wartiovaara, Kirmo; Otonkoski, Timo

    2015-01-01

    Summary CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable dCas9 activator version by fusion with the dihydrofolate reductase (DHFR) destabilization domain. Finally, we show that the destabilized dCas9 activator can be used to control human pluripotent stem cell differentiation into endodermal lineages. PMID:26352799

  19. Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system.

    PubMed

    Anton, Tobias; Bultmann, Sebastian; Leonhardt, Heinrich; Markaki, Yolanda

    2014-01-01

    Labeling and tracing of specific sequences in living cells has been a major challenge in studying the spatiotemporal dynamics of native chromatin. Here we repurposed the prokaryotic CRISPR/Cas adaptive immunity system to specifically detect endogenous genomic loci in mouse embryonic stem cells. We constructed a catalytically inactive version of the Cas9 endonuclease, fused it with eGFP (dCas9-eGFP) and co-expressed small guide RNAs (gRNAs) to target pericentric, centric, and telomeric repeats, which are enriched in distinct nuclear structures. With major satellite specific gRNAs we obtained a characteristic chromocenter (CC) pattern, while gRNAs targeting minor satellites and telomeres highlighted smaller foci coinciding with centromere protein B (CENP-B) and telomeric repeat-binding factor 2 (TRF2), respectively. DNA sequence specific labeling by gRNA/dCas9-eGFP complexes was directly shown with 3D-fluorescent in situ hybridization (3D-FISH). Structured illumination microscopy (3D-SIM) of gRNA/dCas9-eGFP expressing cells revealed chromatin ultrastructures and demonstrated the potential of this approach for chromatin conformation studies by super resolution microscopy. This programmable dCas9 labeling system opens new perspectives to study functional nuclear architecture. PMID:24637835

  20. Application of the genome editing tool CRISPR/Cas9 in non-human primates

    PubMed Central

    LUO, Xin; LI, Min; SU, Bing

    2016-01-01

    In the past three years, RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system has been used to facilitate efficient genome editing in many model and non-model animals. However, its application in nonhuman primates is still at the early stage, though in view of the similarities in anatomy, physiology, behavior and genetics, closely related nonhuman primates serve as optimal models for human biology and disease studies. In this review, we summarize the current proceedings of gene editing using CRISPR/Cas9 in nonhuman primates. PMID:27469252

  1. Applications of the CRISPR-Cas9 system in cancer biology.

    PubMed

    Sánchez-Rivera, Francisco J; Jacks, Tyler

    2015-07-01

    The prokaryotic type II CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system is rapidly revolutionizing the field of genetic engineering, allowing researchers to alter the genomes of a large range of organisms with relative ease. Experimental approaches based on this versatile technology have the potential to transform the field of cancer genetics. Here, we review current approaches for functional studies of cancer genes that are based on CRISPR-Cas, with emphasis on their applicability for the development of next-generation models of human cancer. PMID:26040603

  2. Applications of the CRISPR-Cas9 system in cancer biology

    PubMed Central

    Sánchez-Rivera, Francisco J.; Jacks, Tyler

    2015-01-01

    Preface The prokaryotic type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is rapidly revolutionizing the field of genetic engineering, allowing researchers to alter the genomes of a large variety of organisms with relative ease. Experimental approaches based on this versatile technology have the potential to transform the field of cancer genetics. Here we review current approaches based on CRISPR-Cas9 for functional studies of cancer genes, with emphasis on its applicability for the development of the next-generation models of human cancer. PMID:26040603

  3. Closure Report for Corrective Action Unit 262: Area 25 Septic Systems and Underground Discharge Point, Nevada Test Site, Nevada

    SciTech Connect

    D. S. Tobiason

    2003-07-01

    This Closure Report (CR) documents the activities undertaken to close Corrective Action Unit (CAU) 262: Area 25 Septic Systems and Underground Discharge Point, in accordance with the Federal Facility Agreement and Consent Order (FFACO) of 1996. Site closure was performed in accordance with the Nevada Division of Environmental Protection (NDEP)-approved Corrective Action Plan (CAP) for CAU 262 (U.S. Department of Energy, National Nuclear Security Administration Nevada Operations Office [NNSA/NV, 2002a]). CAU 262 is located at the Nevada Test Site (NTS) approximately 105 kilometers (65 miles) northwest of Las Vegas, Nevada. CAU 262 consists of the following nine Corrective Action Sites (CASs) located in Area 25 of the NTS: CAS 25-02-06, Underground Storage tank CAS 25-04-06, Septic Systems A and B CAS 25-04-07, Septic System CAS 25-05-03, Leachfield CAS 25-05-05, Leachfield CAS 25-05-06, Leachfield CAS 25-05-08, Radioactive Leachfield CAS 25-05-12, Leachfield CAS 25-51-01, Dry Well.

  4. Closure Report for Corrective Action Unit 562: Waste Systems, Nevada National Security Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2012-08-15

    This Closure Report (CR) presents information supporting closure of Corrective Action Unit (CAU) 562, Waste Systems, and provides documentation supporting the completed corrective actions and confirmation that closure objectives for CAU 562 were met. This CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; the U.S. Department of Energy (DOE), Environmental Management; the U.S. Department of Defense; and DOE, Legacy Management (FFACO, 1996 as amended). CAU 562 consists of the following 13 Corrective Action Sites (CASs), located in Areas 2, 23, and 25 of the Nevada National Security Site: · CAS 02-26-11, Lead Shot · CAS 02-44-02, Paint Spills and French Drain · CAS 02-59-01, Septic System · CAS 02-60-01, Concrete Drain · CAS 02-60-02, French Drain · CAS 02-60-03, Steam Cleaning Drain · CAS 02-60-04, French Drain · CAS 02-60-05, French Drain · CAS 02-60-06, French Drain · CAS 02-60-07, French Drain · CAS 23-60-01, Mud Trap Drain and Outfall · CAS 23-99-06, Grease Trap · CAS 25-60-04, Building 3123 Outfalls Closure activities began in October 2011 and were completed in April 2012. Activities were conducted according to the Corrective Action Plan for CAU 562 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2011). The corrective actions included No Further Action and Clean Closure. Closure activities generated sanitary waste and hazardous waste. Some wastes exceeded land disposal limits and required offsite treatment prior to disposal. Other wastes met land disposal restrictions and were disposed in appropriate onsite or offsite landfills. NNSA/NSO requests the following: · A Notice of Completion from the Nevada Division of Environmental Protection to NNSA/NSO for closure of CAU 562 · The transfer of CAU 562 from Appendix III to Appendix IV, Closed Corrective Action Units, of the FFACO

  5. The Influence of the CAS Standards on Academic Advisors and Advising Programs

    ERIC Educational Resources Information Center

    Keeling, Sarah

    2010-01-01

    Research on the influence of the Council for the Advancement of Standards in Higher Education (CAS) on academic advising is limited. Using a comparative case study method, I respond to this research gap by exploring how the standards influence practices of academic advising programs. Study results indicate that participating advisors knew little…

  6. Enhancing local action planning through quantitative flood risk analysis: a case study in Spain

    NASA Astrophysics Data System (ADS)

    Castillo-Rodríguez, Jesica Tamara; Escuder-Bueno, Ignacio; Perales-Momparler, Sara; Ramón Porta-Sancho, Juan

    2016-07-01

    This article presents a method to incorporate and promote quantitative risk analysis to support local action planning against flooding. The proposed approach aims to provide a framework for local flood risk analysis, combining hazard mapping with vulnerability data to quantify risk in terms of expected annual affected population, potential injuries, number of fatalities, and economic damages. Flood risk is estimated combining GIS data of loads, system response, and consequences and using event tree modelling for risk calculation. The study area is the city of Oliva, located on the eastern coast of Spain. Results from risk modelling have been used to inform local action planning and to assess the benefits of structural and non-structural risk reduction measures. Results show the potential impact on risk reduction of flood defences and improved warning communication schemes through local action planning: societal flood risk (in terms of annual expected affected population) would be reduced up to 51 % by combining both structural and non-structural measures. In addition, the effect of seasonal population variability is analysed (annual expected affected population ranges from 82 to 107 %, compared with the current situation, depending on occupancy rates in hotels and campsites). Results highlight the need for robust and standardized methods for urban flood risk analysis replicability at regional and national scale.

  7. Experimental study on bubble pulse features under the combined action of horizontal and vertical walls

    NASA Astrophysics Data System (ADS)

    Wang, Shi-ping; Chu, Wen-hua; Zhang, A.-man

    2014-06-01

    The pulse features of a bubble have a close connection with the boundary condition. When a bubble moves near a rigid wall, it will be attracted by the Bjerknes force of the wall, and a jet pointing at the wall will be generated. In real application, the bubble may move under the combined action of walls in different directions when it forms at the corner of a pipe or at the bottom of a dam. The motion of the bubble shows complex and nonlinear characteristics under these conditions. In order to investigate the bubble pulse features near complex walls, a horizontal wall and a vertical wall are put into the experimental water tank synchronously, and an electric circuit with 200 voltages is designed to generate discharge bubbles, and then experimental study on the bubble pulse features under the combined action of horizontal and vertical walls is carried out. The influences of the combined action of two walls on the bubble shape, pulse period, moving trace and inside jet are obtained by changing the distances from bubble center to the two walls. It aims at providing references for the relevant theoretical and numerical research.

  8. Affordance effects in grasping actions for graspable objects: electromyographic reaction time study.

    PubMed

    Suzuki, Tomotaka; Takagi, Mineko; Sugawara, Kenichi

    2012-12-01

    It is unclear whether affordance effects shorten the reaction time in the interaction between objects and actions. This study investigated affordance effects based on compatibility between perception of graspable objects and the act of grasping. The electromyographic reaction time (EMG-RT) was used as the response, and Go/NoGo (Experiment 1) and choice (Experiment 2) reaction-time tasks were performed using combinations of two types of stimulus image (tools and animals) and two types of response task (flexion and extension of all fingers). In Experiment 1, no interaction of stimulus images and response tasks occurred, but the EMG-RT for tools was statistically significantly delayed longer than that for animals. In Experiment 2, the EMG-RT of flexion of all fingers for tools was statistically significantly delayed compared with that for animals, showing interaction. Affordance effects based on compatibility of objects and actions are the basis on human-tool interaction. This interaction induces a goal-directed act and prolongs motor execution of grasping actions for them. PMID:23409599

  9. In vitro and in vivo studies of the Interferon-alpha action on distinct Orthobunyavirus.

    PubMed

    Livonesi, Márcia Cristina; de Sousa, Ricardo Luiz Moro; Badra, Soraya Jabur; Figueiredo, Luiz Tadeu Moraes

    2007-08-01

    Oropouche, Caraparu, Guama, Guaroa and Tacaiuma viruses (Orthobunyavirus genus) cause human febrile illnesses and/or encephalitis. To achieve a therapeutical agent to prevent and/or treat these diseases we evaluated the antiviral action of Interferon-alpha (IFN-alpha) on these orthobunyaviruses. In vitro results showed that all the studied orthobunyaviruses are susceptible to antiviral action of IFN-alpha, but this susceptibility is limited and dependent on both concentration of drug and treatment period. In vivo results demonstrated that IFN-alpha present antiviral action on Oropouche and Guaroa viruses when used as a prophylactic treatment. Moreover, a treatment initiated 3h after infection prevented the death of Guaroa virus infected-mice. Additionally, mortality of mice was related to the migration and replication of viruses in their brains. Our results suggest that IFN-alpha could be potentially useful in the prevention of diseases caused by Oropouche virus and in the prevention and/or treatment of diseases caused by Guaroa virus. PMID:17368573

  10. Data Integrity-A Study of Current Regulatory Thinking and Action.

    PubMed

    Shafiei, Nader; De Montardy, Regis; Rivera-Martinez, Edwin

    2015-01-01

    In reaction to breaches of data integrity in the pharmaceutical industry, regulatory authorities have introduced inspection approaches or initiatives with the aim of reducing occurrences of data integrity problems. This review article-based on study of 65 cases of regulatory action from 2002 to 2014-provides an overview of current regulatory thinking and action on breaches of data integrity affecting GxP (health-related regulations) processes supporting non-clinical studies, clinical studies, laboratory controls, and production controls. These case studies largely represent position of the U.S. Food and Drug Administration and the regulatory agencies affiliated with the European Medicines Agency. Also discussed is the role of human factors as a potential source of data integrity problems. The article concludes by recommending some remedial controls that could be established to avoid or reduce occurrences of data integrity problems.Lay Abstract: In fulfilling their mission to protect public health, regulatory agencies (e.g., U.S. Food and Drug Administration, European Medicines Agency) must establish confidence that medical products they approve are fit for their intended use. In so doing they rely on scientific and operational data generated during research, development, manufacturing, sales, marketing, distribution, and post-marketing surveillance activities. The level of confidence they build is directly proportional to the scientific validity and integrity of data presented to them by the sponsors of medical products. In this article we present analysis of 65 case studies that document regulatory action taken by various regulatory agencies on breach of data integrity between 2002 and 2014. The ensuing discussion on current trends largely represents position of the U.S. Food and Drug Administration and European Medicines Agency. The article concludes by proposing some remedial controls that could be established by pharmaceutical companies to avoid or reduce

  11. Judgment of actions in experts: a high-resolution EEG study in elite athletes.

    PubMed

    Babiloni, Claudio; Del Percio, Claudio; Rossini, Paolo M; Marzano, Nicola; Iacoboni, Marco; Infarinato, Francesco; Lizio, Roberta; Piazza, Marina; Pirritano, Mirella; Berlutti, Giovanna; Cibelli, Giuseppe; Eusebi, Fabrizio

    2009-04-01

    The present study tested the two following hypotheses: (i) compared to non-athletes, elite athletes are characterized by a reduced cortical activation during the judgment of sporting observed actions; (ii) in elite athletes, a good judgment of observed sporting actions is related to a low cortical activation. To address these issues, electroencephalographic (EEG) data were recorded in 15 elite rhythmic gymnasts and 13 non-gymnasts. They observed a series of 120 rhythmic gymnastic videos. At the end of each video, the subjects had to judge the artistic/athletic level of the exercise by a scale from 0 to 10. The mismatch between their judgment and that of the coach indexed the degree of action judgment. The EEG cortical sources were estimated by sLORETA. With reference to a pre-stimulus period, the power decrease of alpha (8-12 Hz) rhythms during the videos indexed the cortical activation (event related desynchronization, ERD). Regarding the hypothesis (i), low- and high-frequency alpha ERD was lower in amplitude in the elite rhythmic gymnasts compared to the non-gymnasts in occipital and temporal areas (ventral pathway) and in dorsal pathway. Regarding the hypothesis (ii), in the elite rhythmic gymnasts high-frequency alpha ERD was higher in amplitude with the videos characterized by a high judgment error than those characterized by a low judgment error; this was true in inferior posterior parietal and ventral premotor areas ("mirror" pathway). These results globally suggest that the judgment of observed sporting actions is related to low amplitude of alpha ERD, as a possible index of spatially selective cortical activation ("neural efficiency"). PMID:19111623

  12. Action-Monitoring Dysfunction in Obstructive Sleep Apnea - A Pilot Study

    PubMed Central

    Chou, Ping-Song; Hsu, Chung-Yao; Wu, Meng-Ni; Liou, Li-Min; Lu, Shinag-Ru; Liu, Ching-Kuan; Lai, Chiou-Lian

    2016-01-01

    Obstructive sleep apnea (OSA) is associated with a broad range of frontal lobe dysfunctions. However, no study has investigated action monitoring, a crucial domain of frontal cognitive functions, in patients with OSA. By using the modified Flanker task, we tested the hypothesis that patients with OSA have an impaired action monitoring function. We recruited 25 untreated patients with moderate–severe OSA and 12 control participants who were matched for age, sex, apolipoprotein E4, and education level. Every enrolled participant underwent a standard overnight laboratory-based polysomnography and completed a modified Flanker task. Compared with the controls, the patients with OSA presented a significantly lower correct response rate in all trials (78.9% vs 95.9%, P = .008), congruent trials (84.7% vs 98.3%, P = .016), and incongruent trials (77.4% vs 94.7%, P = .009). The post-error correction rate was significantly lower in the patients with OSA than in the controls (74.9% vs 93.8%, P = .005). Furthermore, strong significant correlations were observed between the arousal index and correct rate in all trials (r = −0.390, P < .05) and in the incongruent trials (r = −0.429, P < .01), as well as between the arousal index and rate of post-error correction (r = −0.435, P < .01). We concluded that the action monitoring function was impaired in the patients with OSA. Sleep fragmentation was a major determinant of impaired action monitoring in these patients. PMID:27300504

  13. Less irritative action of wine and Japanese sake in rat stomachs: a comparative study with ethanol.

    PubMed

    Nakagiri, Akari; Fukushima, Kazuhiro; Kato, Shinichi; Takeuchi, Koji

    2006-02-01

    The ingestion of alcohol, especially in excess, causes acute gastric lesions and gastritis in humans, yet the mucosal irritative action of alcoholic beverages remains largely unknown. We examined the mucosal irritative action of whiskey, wine and Japanese sake in the rat stomach both ex vivo and in vitro, in comparison with ethanol. Under urethane anesthesia, a rat stomach was mounted in an ex vivo chamber, then superfused with saline, and the transmucosal potential difference (PD) was measured. After the basal PD had stabilized, the mucosa was exposed for 30 min to 2 ml of 15% ethanol, whiskey (containing 15% ethanol), white wine, or Japanese sake (the ethanol concentration of the latter two is 12-15%). In the in vitro study, rat epithelial cells (RGM1) were treated with the alcoholic beverages for 5 min, and the cell viability was determined with crystal violet. Ethanol or whiskey applied to the chamber caused a decrease in PD, while wine or Japanese sake did not. Histologically, surface epithelial damage was observed after exposure to both ethanol and whiskey, yet no damage was induced by white wine and Japanese sake. Likewise, both ethanol and whiskey markedly reduced the viability of RGM1 cells after 5 min of incubation, while neither white wine nor Japanese sake had any effect. In addition, supplementation of glucose significantly prevented the reduction in both PD and cell viability caused by ethanol. These results suggest that the mucosal irritative action of Japanese sake and white wine is much less pronounced than that of ethanol or whiskey and that the less damaging action of Japanese sake and white wine may be, at least partly, accounted for by the glucose contained in these alcoholic beverages. PMID:16534671

  14. Closure Report for Corrective Action Unit 548: Areas 9, 10, 18, 19, and 20 Housekeeping Sites, Nevada National Security Site, Nevada

    SciTech Connect

    NSTec Environmental Restoration

    2012-08-27

    This Closure Report (CR) documents closure activities for Corrective Action Unit (CAU) 548, Areas 9, 10, 18, 19, and 20 Housekeeping Sites, and complies with the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; the U.S. Department of Energy (DOE), Environmental Management; the U.S. Department of Defense; and DOE, Legacy Management (FFACO, 1996 as amended). CAU 548 consists of the following Corrective Action Sites (CASs), located in Areas 9, 10, 12, 18, 19, and 20 of the Nevada National Security Site: · CAS 09-99-02, Material Piles (2) · CAS 09-99-04, Wax, Paraffin · CAS 09-99-05, Asbestos, Vermiculite · CAS 09-99-07, Tar Spill · CAS 10-22-02, Drums · CAS 10-22-05, Gas Block · CAS 10-22-07, Gas Block · CAS 10-22-34, Drum · CAS 10-22-38, Drum; Cable · CAS 12-99-04, Epoxy Tar Spill · CAS 12-99-08, Cement Spill · CAS 18-14-01, Transformers (3) · CAS 19-22-01, Drums · CAS 19-22-11, Gas Block (2) · CAS 19-44-01, Fuel Spill · CAS 20-22-07, Drums (2) · CAS 20-22-09, Drums (3) · CAS 20-22-14, Drums (2) · CAS 20-22-16, Drums (2) · CAS 20-24-09, Battery Closure activities began in July 2011 and were completed in December 2011 and included removal and disposal of material piles, spills, sanitary debris, a lead acid battery, lead and steel shot, and stained soil. Activities were conducted according to the Sectored Clean-up Work Plan for Housekeeping Category Waste Sites (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2003). Closure activities generated sanitary waste, hydrocarbon waste, low-level waste, hazardous waste, and mixed waste. Some wastes exceeded land disposal limits and required offsite treatment prior to disposal. Other wastes met land disposal restrictions and were disposed in appropriate onsite or offsite landfills. NNSA/NSO requests the following: · A Notice of Completion from the Nevada Division of Environmental Protection to NNSA/NSO for

  15. Action Research as another Literacy Skill To Improve Academic Performance: A Case Study of Empowered Language Learning

    ERIC Educational Resources Information Center

    Ariizumi, Yoshihiko

    2006-01-01

    Background: Even though action research has been employed in an increasing number of fields of practice including teaching, nursing, and business, application in learning has been scarcely reported. This case study is one of the first attempts to apply action research principles to help learners improve their learning performance. Purpose: This…

  16. An Exploratory Study of Undergraduates' Attitudes toward Affirmative Action Policies for Asian Americans in College

    ERIC Educational Resources Information Center

    Hartlep, Nicholas Daniel; Lowinger, Robert Jay

    2014-01-01

    This exploratory study examined white undergraduate students' (a) racial attitudes towards Asian Americans, (b) principled policy attitudes toward affirmative action, and (c) self-interest in relation to their support for college-based affirmative action policies for Asian Americans at a Midwestern university. A sample (n = 264, 28% male, 72%…

  17. Using Reflective Practice to Incorporate Formative Assessment in a Middle School Science Classroom: A Participatory Action Research Study

    ERIC Educational Resources Information Center

    Trauth-Nare, Amy; Buck, Gayle

    2011-01-01

    Our purpose was to investigate the efficacy of using reflective practice to guide our action research study of incorporating formative assessment into middle school science teaching and learning. Using participatory action research, we worked collaboratively to incorporate formative assessment into two instructional units, and then engaged in…

  18. Cas5d processes pre-crRNA and is a member of a larger family of CRISPR RNA endonucleases

    PubMed Central

    Garside, Erin L.; Schellenberg, Matthew J.; Gesner, Emily M.; Bonanno, Jeffrey B.; Sauder, J. Michael; Burley, Stephen K.; Almo, Steven C.; Mehta, Garima; MacMillan, Andrew M.

    2012-01-01

    Small RNAs derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci in bacteria and archaea are involved in an adaptable and heritable gene-silencing pathway. Resistance to invasive genetic material is conferred by the incorporation of short DNA sequences derived from this material into the genome as CRISPR spacer elements separated by short repeat sequences. Processing of long primary transcripts (pre-crRNAs) containing these repeats by a CRISPR-associated (Cas) RNA endonuclease generates the mature effector RNAs that target foreign nucleic acid for degradation. Here we describe functional studies of a Cas5d ortholog, and high-resolution structural studies of a second Cas5d family member, demonstrating that Cas5d is a sequence-specific RNA endonuclease that cleaves CRISPR repeats and is thus responsible for processing of pre-crRNA. Analysis of the structural homology of Cas5d with the previously characterized Cse3 protein allows us to model the interaction of Cas5d with its RNA substrate and conclude that it is a member of a larger family of CRISPR RNA endonucleases. PMID:23006625

  19. Toxicology and carcinogenesis studies of diethylphthalate (Cas No. 84-66-2) in F344/n rats and B6C3F1 mice (dermal studies) with dermal initiation/promotion study of diethylphthalate and dimethylphthalate (Cas No. 131-11-3) in male Swiss (CD-1 (trade name)) mice. Technical report

    SciTech Connect

    1995-05-01

    Toxicology and carcinogenicity studies were conducted by dermal administration of diethylphthalate to groups of 60 F344/N rats of each sex at doses of 0, 100, or 300 microL and to groups of 60 B6C3F1 mice of each sex at doses of 0, 7.5, 15, or 30 microL. Neat chemical was applied to rats for 5 days per week for 103 weeks and up to 10 animals per group were evaluated after 15 months. Mice received doses in 100 microL of acetone for 5 days per weeks for 103 weeks with a 1 week recovery period, and up to 10 animals per group were evaluated after 15 months. Under the conditions of these 2-year dermal studies, there was not evidence of carcinogenic activity of diethylphthalate in male or female F344/N rats receiving 100 or 300 microL. There was equivocal evidence of carcinogenic activity of diethylphthalate in male and female B6C3F1 mice based on increased incidences of hepatocellular neoplasms, primarily adenomas. In the initiation/promotion model, there was no evidence of initiating or promoting activity of diethylphthalate or dimethylphthalate in male Swiss (CD-1) mice.

  20. 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

  1. Targeted mutagenesis in soybean using the CRISPR-Cas9 system.

    PubMed

    Sun, Xianjun; Hu, Zheng; Chen, Rui; Jiang, Qiyang; Song, Guohua; Zhang, Hui; Xi, Yajun

    2015-01-01

    Genome editing is a valuable technique for gene function analysis and crop improvement. Over the past two years, the CRISPR-Cas9 system has emerged as a powerful tool for precisely targeted gene editing. In this study, we predicted 11 U6 genes in soybean (Glycine max L.). We then constructed two vectors (pCas9-GmU6-sgRNA and pCas9-AtU6-sgRNA) using the soybean U6-10 and Arabidopsis U6-26 promoters, respectively, to produce synthetic guide RNAs (sgRNAs) for targeted gene mutagenesis. Three genes, Glyma06g14180, Glyma08g02290 and Glyma12g37050, were selected as targets. Mutations of these three genes were detected in soybean protoplasts. The vectors were then transformed into soybean hairy roots by Agrobacterium rhizogenes infection, resulting in efficient target gene editing. Mutation efficiencies ranged from 3.2-9.7% using the pCas9-AtU6-sgRNA vector and 14.7-20.2% with the pCas9-GmU6-sgRNA vector. Biallelic mutations in Glyma06g14180 and Glyma08g02290 were detected in transgenic hairy roots. Off-target activities associated with Glyma06g14180 and Glyma12g37050 were also detected. Off-target activity would improve mutation efficiency for the construction of a saturated gene mutation library in soybean. Targeted mutagenesis using the CRISPR-Cas9 system should advance soybean functional genomic research, especially that of genes involved in the roots and nodules. PMID:26022141

  2. CRISPR-Cas9-Mediated Single-Gene and Gene Family Disruption in Trypanosoma cruzi

    PubMed Central

    Peng, Duo; Kurup, Samarchith P.; Yao, Phil Y.; Minning, Todd A.

    2014-01-01

    ABSTRACT Trypanosoma cruzi is a protozoan parasite of humans and animals, affecting 10 to 20 million people and innumerable animals, primarily in the Americas. Despite being the largest cause of infection-induced heart disease worldwide, even among the neglected tropical diseases (NTDs) T. cruzi is considered one of the least well understood and understudied. The genetic complexity of T. cruzi as well as the limited set of efficient techniques for genome engineering contribute significantly to the relative lack of progress in and understanding of this pathogen. Here, we adapted the CRISPR-Cas9 system for the genetic engineering of T. cruzi, demonstrating rapid and efficient knockout of multiple endogenous genes, including essential genes. We observed that in the absence of a template, repair of the Cas9-induced double-stranded breaks (DSBs) in T. cruzi occurs exclusively by microhomology-mediated end joining (MMEJ) with various-sized deletions. When a template for DNA repair is provided, DSB repair by homologous recombination is achieved at an efficiency several orders of magnitude higher than that in the absence of CRISPR-Cas9-induced DSBs. We also demonstrate the high multiplexing capacity of CRISPR-Cas9 in T. cruzi by knocking down expression of an enzyme gene family consisting of 65 members, resulting in a significant reduction of enzymatic product with no apparent off-target mutations. Lastly, we show that Cas9 can mediate disruption of its own coding sequence, rescuing a growth defect in stable Cas9-expressing parasites. These results establish a powerful new tool for the analysis of gene functions in T. cruzi, enabling the study of essential genes and their functions and analysis of the many large families of related genes that occupy a substantial portion of the T. cruzi genome. PMID:25550322

  3. Evaporated CaS thin films for AC electroluminescence devices

    NASA Astrophysics Data System (ADS)

    Kobayashi, H.; Tanaka, S.; Shanker, V.; Shiiki, M.; Deguchi, H.

    1985-08-01

    The growth behavior of evaporated CaS thin films has been investigated to achieve bright electroluminescence. The crystallinity of CaS films is improved with substrate temperature and for temperatures higher than 300°C, the films orient to the (200) plane. Sulfur coevaporation further helps to form a more perfect film even at lower temperatures. A CaS: Ce,Cl electroluminescent thin film device has been fabricated with a brightness of 650 cd/m 2.

  4. Polychlorinated biphenyls in the electrical machinery industry: an ethnological study of community action and corporate responsibility.

    PubMed

    Nash, J; Kirsch, M

    1986-01-01

    Environmental and occupational health problems cannot be understood through purely medical and epidemiological analyses, the social forces affecting biologically adaptive behavior must also be analyzed. Research on the political economy of health needs to generate an ethnology of community action relevant to the analyses of corporate structures for which it is best known. In studying the community of Pittsfield, Mass., where a General Electric plant is located, we encountered environmental and occupational health problems in just this context. This essay is, therefore, an effort to extend the political economy of health into the ethnological domain of community research. PMID:3092365

  5. 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

  6. 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. PMID:26503043

  7. Cas9 as a versatile tool for engineering biology

    PubMed Central

    Mali, Prashant; Esvelt, Kevin M; Church, George M

    2014-01-01

    RNA-guided Cas9 nucleases derived from clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have dramatically transformed our ability to edit the genomes of diverse organisms. We believe tools and techniques based on Cas9, a single unifying factor capable of colocalizing RNA, DNA and protein, will grant unprecedented control over cellular organization, regulation and behavior. Here we describe the Cas9 targeting methodology, detail current and prospective engineering advances and suggest potential applications ranging from basic science to the clinic. PMID:24076990

  8. Optical Control of CRISPR/Cas9 Gene Editing

    PubMed Central

    Hemphill, James; Borchardt, Erin K.; Brown, Kalyn; Asokan, Aravind; Deiters, Alexander

    2016-01-01

    The CRISPR/Cas9 system has emerged as an important tool in biomedical research for a wide range of applications, with significant potential for genome engineering and gene therapy. In order to achieve conditional control of the CRISPR/Cas9 system, a genetically encoded light-activated Cas9 was engineered through the site-specific installation of a caged lysine amino acid. Several potential lysine residues were identified as viable caging sites that can be modified to optically control Cas9 function, as demonstrated through optical activation and deactivation of both exogenous and endogenous gene function. PMID:25905628

  9. DNA targeting by the type I-G and type I-A CRISPR–Cas systems of Pyrococcus furiosus

    PubMed Central

    Elmore, Joshua; Deighan, Trace; Westpheling, Jan; Terns, Rebecca M.; Terns, Michael P.

    2015-01-01

    CRISPR–Cas systems silence plasmids and viruses in prokaryotes. CRISPR–Cas effector complexes contain CRISPR RNAs (crRNAs) that include sequences captured from invaders and direct CRISPR-associated (Cas) proteins to destroy corresponding invader nucleic acids. Pyrococcus furiosus (Pfu) harbors three CRISPR–Cas immune systems: a Cst (Type I-G) system with an associated Cmr (Type III-B) module at one locus, and a partial Csa (Type I-A) module (lacking known invader sequence acquisition and crRNA processing genes) at another locus. The Pfu Cmr complex cleaves complementary target RNAs, and Csa systems have been shown to target DNA, while the mechanism by which Cst complexes silence invaders is unknown. In this study, we investigated the function of the Cst as well as Csa system in Pfu strains harboring a single CRISPR–Cas system. Plasmid transformation assays revealed that the Cst and Csa systems both function by DNA silencing and utilize similar flanking sequence information (PAMs) to identify invader DNA. Silencing by each system specifically requires its associated Cas3 nuclease. crRNAs from the 7 shared CRISPR loci in Pfu are processed for use by all 3 effector complexes, and Northern analysis revealed that individual effector complexes dictate the profile of mature crRNA species that is generated. PMID:26519471

  10. Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus.

    PubMed

    Arazoe, Takayuki; Miyoshi, Kennosuke; Yamato, Tohru; Ogawa, Tetsuo; Ohsato, Shuichi; Arie, Tsutomu; Kuwata, Shigeru

    2015-12-01

    CRISPR/Cas-derived RNA-guided nucleases (RGNs) that can generate DNA double-strand breaks (DSBs) at a specific sequence are widely used for targeted genome editing by induction of DSB repair in many organisms. The CRISPR/Cas system consists of two components: a single Cas9 nuclease and a single-guide RNA (sgRNA). Therefore, the system for constructing RGNs is simple and efficient, but the utilization of RGNs in filamentous fungi has not been validated. In this study, we established the CRISPR/Cas system in the model filamentous fungus, Pyricularia oryzae, using Cas9 that was codon-optimized for filamentous fungi, and the endogenous RNA polymerase (RNAP) III U6 promoter and a RNAP II fungal promoter for the expression of the sgRNA. We further demonstrated that RGNs could recognize the desired sequences and edit endogenous genes through homologous recombination-mediated targeted gene replacement with high efficiency. Our system will open the way for the development of various CRISPR/Cas-based applications in filamentous fungi. PMID:26039904

  11. Neuroplasticity of imagined wrist actions after spinal cord injury: a pilot study.

    PubMed

    Di Rienzo, Franck; Guillot, Aymeric; Mateo, Sébastien; Daligault, Sébastien; Delpuech, Claude; Rode, Gilles; Collet, Christian

    2015-01-01

    Motor imagery (MI - i.e., the mental representation of an action without physically executing it) stimulates brain motor networks and promotes motor learning after spinal cord injury (SCI). An interesting issue is whether the brain networks controlling MI are being reorganized with reference to spared motor functions. In this pilot study, we tested using magnetoencephalography (MEG) whether changes in cortical recruitment during MI were related to the motor changes elicited by rehabilitation. Over a 1-year period of inclusion, C6 SCI participants (n = 4) met stringent criteria for inclusion in a rehabilitation program focused on the tenodesis prehension (i.e., a compensatory prehension enabling seizing of objects in spite of hand and forearm muscles paralysis). After an extended baseline period of 5 weeks including repeated MEG and chronometric assessments of motor performance, MI training was embedded to the classical course of physiotherapy for five additional weeks. Posttest MEG and motor performance data were collected. A group of matched healthy control participants underwent a similar procedure. The MI intervention resulted in changes in the variability of the wrist extensions, i.e., a key movement of the tenodesis grasp (p < .05). Interestingly, the extent of cortical recruitment, quantified by the number of MEG activation sources recorded within Brodmann areas 1-8 during MI of the wrist extension, significantly predicted actual movement variability changes across sessions (p < .001). However, no such relationship was present for movement times. Repeated measurements afforded a reliable statistical power (range .70-.97). This pilot study does not provide straightforward evidence of MI efficacy, which would require a randomized controlled trial. Nonetheless, the data showed that the relationship between action and imagery of spared actions may be preserved after SCI. PMID:25300960

  12. The Impact of CAS Standards on the Practice of Student Affairs.

    ERIC Educational Resources Information Center

    Mann, Barbara A.; And Others

    1991-01-01

    Studied impact of Council for the Advancement of Standards (CAS) for Student Services/Development Programs'"Standards and Guidelines for Student Services/Development Programs." Findings from 130 chief student affairs officers revealed that, although emphasis on assessment, student involvement, and holistic development suggested by standards was…

  13. Consensus of Chief Student Affairs Officers toward the CAS Orientation Standards.

    ERIC Educational Resources Information Center

    Nadler, Daniel P.; Miller, Michael T.

    The role of the chief student affairs officer (CSAO) has become paramount to the stability of colleges and universities. Since the CSAO typically determines the direction and framework for the student affairs program, the purpose of this study was to identify CSAOs' levels of agreement on the Council for the Advancement of Standards (CAS) for…

  14. An Analytical Framework for Categorizing the Use of CAS Symbolic Manipulation in Textbooks

    ERIC Educational Resources Information Center

    Davis, Jon D.; Fonger, Nicole L.

    2015-01-01

    The symbolic manipulation capabilities of computer algebra systems, which we refer to as CAS-S, are now becoming instantiated within secondary mathematics textbooks in the United States for the first time. While a number of research studies have examined how teachers use this technology in their classrooms, one of the most important factors in how…

  15. Examinations in the Final Year of Transition to Mathematical Methods Computer Algebra System (CAS)

    ERIC Educational Resources Information Center

    Leigh-Lancaster, David; Les, Magdalena; Evans, Michael

    2010-01-01

    2009 was the final year of parallel implementation for Mathematical Methods Units 3 and 4 and Mathematical Methods (CAS) Units 3 and 4. From 2006-2009 there was a common technology-free short answer examination that covered the same function, algebra, calculus and probability content for both studies with corresponding expectations for key…

  16. Corrective Action Plan for Corrective Action Unit 261: Area 25 Test Cell A Leachfield System, Nevada Test Site, Nevada

    SciTech Connect

    T. M. Fitzmaurice

    2000-08-01

    This Corrective Action Plan (CAP) has been prepared for the Corrective Action Unit (CAU)261 Area 25 Test Cell A Leachfield System in accordance with the Federal Facility and Consent Order (Nevada Division of Environmental Protection [NDEP] et al., 1996). This CAP provides the methodology for implementing the approved corrective action alternative as listed in the Corrective Action Decision Document (U.S. Department of Energy, Nevada Operations Office, 1999). Investigation of CAU 261 was conducted from February through May of 1999. There were no Constituents of Concern (COCs) identified at Corrective Action Site (CAS) 25-05-07 Acid Waste Leach Pit (AWLP). COCs identified at CAS 25-05-01 included diesel-range organics and radionuclides. The following closure actions will be implemented under this plan: Because COCs were not found at CAS 25-05-07 AWLP, no action is required; Removal of septage from the septic tank (CAS 25-05-01), the distribution box and the septic tank will be filled with grout; Removal of impacted soils identified near the initial outfall area; and Upon completion of this closure activity and approval of the Closure Report by NDEP, administrative controls, use restrictions, and site postings will be used to prevent intrusive activities at the site.

  17. Tenth anniversary of CAS ONLINE service : What CAS services should be in the new era of chemical information

    NASA Astrophysics Data System (ADS)

    Kostakos, Charles N.

    Chemical Abstracts Service celebrated 10th anniversary of CAS online information service in 1990. A speech given on the occasion reviewed history of the CAS ONLINE, in relation to its most important benefits for scientists and engineers. The development of STN international, the network through which CAS ONLINE is accessible around the world, was also discussed in the speech. The CAS ONLINE now contains a wide variety of files relating to chemical field including CA file, Registry file. CA previews,. CASREACT, CIN. MARPAT, etc for supplying chemical information worldwide.

  18. The impact of inquiry-based instructional professional development upon instructional practice: An action research study

    NASA Astrophysics Data System (ADS)

    Broom, Frances A.

    This mixed method case study employs action research, conducted over a three month period with 11 elementary math and science practitioners. Inquiry as an instructional practice is a vital component of math and science instruction and STEM teaching. Teachers examined their beliefs and teaching practices with regard to those instructional factors that influence inquiry instruction. Video-taped lessons were compared to a rubric and pre and post questionnaires along with two interviews which informed the study. The results showed that while most beliefs were maintained, teachers implemented inquiry at a more advanced level after examining their teaching and reflecting on ways to increase inquiry practices. Because instructional practices provide only one component of inquiry-based instruction, other components need to be examined in a future study.

  19. Applying Organizational Theories to Action Research in Community Settings: A Case Study in Urban Schools

    ERIC Educational Resources Information Center

    Bryan, Keli S.; Klein, Dena A.; Elias, Maurice J.

    2007-01-01

    Action research is well grounded in the worlds of organizational and community psychology. The practice of action research within each of these fields has been shaped by their dominant settings, theories, and values; where these diverge, rich learning opportunities have been created. Each phase of the action research cycle has particular…

  20. The Influence of Action Perception on Object Recognition: A Developmental Study

    ERIC Educational Resources Information Center

    Mounoud, Pierre; Duscherer, Katia; Moy, Guenael; Perraudin, Sandrine

    2007-01-01

    Two experiments explored the existence and the development of relations between action representations and object representations. A priming paradigm was used in which participants viewed an action pantomime followed by the picture of a tool, the tool being either associated or unassociated with the preceding action. Overall, we observed that the…

  1. Genomic and Proteomic Studies on the Mode of Action of Oxaboroles against the African Trypanosome

    PubMed Central

    Jones, Deuan C.; Foth, Bernardo J.; Urbaniak, Michael D.; Patterson, Stephen; Ong, Han B.; Berriman, Matthew; Fairlamb, Alan H.

    2015-01-01

    SCYX-7158, an oxaborole, is currently in Phase I clinical trials for the treatment of human African trypanosomiasis. Here we investigate possible modes of action against Trypanosoma brucei using orthogonal chemo-proteomic and genomic approaches. SILAC-based proteomic studies using an oxaborole analogue immobilised onto a resin was used either in competition with a soluble oxaborole or an immobilised inactive control to identify thirteen proteins common to both strategies. Cell-cycle analysis of cells incubated with sub-lethal concentrations of an oxaborole identified a subtle but significant accumulation of G2 and >G2 cells. Given the possibility of compromised DNA fidelity, we investigated long-term exposure of T. brucei to oxaboroles by generating resistant cell lines in vitro. Resistance proved more difficult to generate than for drugs currently used in the field, and in one of our three cell lines was unstable. Whole-genome sequencing of the resistant cell lines revealed single nucleotide polymorphisms in 66 genes and several large-scale genomic aberrations. The absence of a simple consistent mechanism among resistant cell lines and the diverse list of binding partners from the proteomic studies suggest a degree of polypharmacology that should reduce the risk of resistance to this compound class emerging in the field. The combined genetic and chemical biology approaches have provided lists of candidates to be investigated for more detailed information on the mode of action of this promising new drug class. PMID:26684831

  2. Studies on the mechanism of action of benzamide riboside: a novel inhibitor of IMP dehydrogenase.

    PubMed

    Gharehbaghi, Kamran; Grünberger, Werner; Jayaram, Hiremagalur N

    2002-04-01

    Benzamide is a well known inhibitor of poly(ADP-ribose)polymerase, an enzyme involved in DNA repair. However, benzamide exhibited neuotoxicity in animals and hence, in the hope of overcoming this problem, benzamide riboside (BR) was synthesized. Our mechanism of action studies on BR suggested that the agent was being metabolized to its 5'-monophosphate and then to its NAD analogue (BAD, benzamide adenine dinucleotide) that inhibits Inosine 5'-monophosphate dehydrogenase (IMPDH). IMPDH is the rate-limiting enzyme of the branched purine nucleotide synthetic pathway that provides guanylates including GTP and dGTP. There are two isoforms of IMPDH, type I that is constitutively present in all cells, and type II that is inducible and is present in highly proliferating cells such as cancer. Ongoing studies with BR analogues suggest that they are more selective in inhibiting IMPDH type II. Our studies have characterized the metabolites of BR, especially its NAD analogue, BAD, by synthesizing this active metabolite by enzymatic means, and identifying its structure by NMR and mass spectrometry. We have partially purified IMPDH from tumor cells and have examined the kinetics of inhibition of IMPDH by BAD. We have also compared biochemical and cytotoxic activities of BR with tiazofurin and selenazofurin, that share similar mechanisms of action with BR. Our studies demonstrated that 2-3-fold more BAD is formed compared to TAD and SAD, the active metabolites of tiazofurin and selenazofurin, respectively. BR has demonstrated potent cytotoxic activity in a diverse group of human tumor cells, specifically more active in sarcomas and CNS neoplasms compared to tiazofurin or selenazofurin. Future in vivo animal studies should set a stage for determining its effectiveness in clinical Phase I studies. PMID:11966437

  3. Recent Advances in Genome Editing Using CRISPR/Cas9

    PubMed Central

    Ding, Yuduan; Li, Hong; Chen, Ling-Ling; Xie, Kabin

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genome engineering that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing method, summarize the recent advances in CRISPR/Cas9 technology, and discuss their implications for plant research. To date, targeted gene knockout using the Cas9/gRNA system has been established in many plant species, and the targeting efficiency and capacity of Cas9 has been improved by optimizing its expression and that of its gRNA. The CRISPR/Cas9 system can also be used for sequence-specific mutagenesis/integration and transcriptional control of target genes. We also discuss off-target effects and the constraint that the protospacer-adjacent motif (PAM) puts on CRISPR/Cas9 genome engineering. To address these problems, a number of bioinformatic tools are available to help design specific gRNAs, and new Cas9 variants and orthologs with high fidelity and alternative PAM specificities have been engineered. Owing to these recent efforts, the CRISPR/Cas9 system is becoming a revolutionary and flexible tool for genome engineering. Adoption of the CRISPR/Cas9 technology in plant research would enable the investigation of plant biology at an unprecedented depth and create innovative applications in precise crop breeding. PMID:27252719

  4. Involvement of the Motor System in Comprehension of Non-Literal Action Language: A Meta-Analysis Study.

    PubMed

    Yang, Jie; Shu, Hua

    2016-01-01

    Although numerous studies have shown that the sensory-motor system is involved in semantic processing of language stimuli, it is still unclear whether comprehension of abstract concepts is embodied, and whether the involvement of the sensory-motor system is context-dependent. Investigation of how the motor system is activated during comprehension of non-literal action languages can help address these issues. So far several studies have reported brain activations during non-literal action language comprehension, but the findings are highly inconsistent because of different types of non-literal action language stimuli. To clarify how the motor system is involved in comprehension of different types of non-literal languages, the current study conducted quantitative meta-analyses on fMRI findings about comprehension of sentences describing fictive motions, metaphoric actions, and idiomatic actions. Results showed that fictive motion sentences elicited activation in the right parahippocampal gyrus, an area important for spatial processing. For metaphoric actions, the left precentral gyrus (BA 6) was strongly activated, suggesting a link between metaphoric and literal meanings. For idiomatic actions, activity was found in the left inferior frontal gyrus (BA 44/45), highlighting semantic selection and inhibition. No premotor or motor activity was found in idiom condition. These results together suggest that the involvement of the sensory-motor system in abstract concepts processing is flexible, depending on semantic features of the language stimuli and links between abstract and literal meanings. PMID:25681159

  5. Factors Affecting Collective Action for Forest Fire Management: A Comparative Study of Community Forest User Groups in Central Siwalik, Nepal

    NASA Astrophysics Data System (ADS)

    Sapkota, Lok Mani; Shrestha, Rajendra Prasad; Jourdain, Damien; Shivakoti, Ganesh P.

    2015-01-01

    The attributes of social ecological systems affect the management of commons. Strengthening and enhancing social capital and the enforcement of rules and sanctions aid in the collective action of communities in forest fire management. Using a set of variables drawn from previous studies on the management of commons, we conducted a study across 20 community forest user groups in Central Siwalik, Nepal, by dividing the groups into two categories based on the type and level of their forest fire management response. Our study shows that the collective action in forest fire management is consistent with the collective actions in other community development activities. However, the effectiveness of collective action is primarily dependent on the complex interaction of various variables. We found that strong social capital, strong enforcement of rules and sanctions, and users' participation in crafting the rules were the major variables that strengthen collective action in forest fire management. Conversely, users' dependency on a daily wage and a lack of transparency were the variables that weaken collective action. In fire-prone forests such as the Siwalik, our results indicate that strengthening social capital and forming and enforcing forest fire management rules are important variables that encourage people to engage in collective action in fire management.

  6. Factors affecting collective action for forest fire management: a comparative study of community forest user groups in central Siwalik, Nepal.

    PubMed

    Sapkota, Lok Mani; Shrestha, Rajendra Prasad; Jourdain, Damien; Shivakoti, Ganesh P

    2015-01-01

    The attributes of social ecological systems affect the management of commons. Strengthening and enhancing social capital and the enforcement of rules and sanctions aid in the collective action of communities in forest fire management. Using a set of variables drawn from previous studies on the management of commons, we conducted a study across 20 community forest user groups in Central Siwalik, Nepal, by dividing the groups into two categories based on the type and level of their forest fire management response. Our study shows that the collective action in forest fire management is consistent with the collective actions in other community development activities. However, the effectiveness of collective action is primarily dependent on the complex interaction of various variables. We found that strong social capital, strong enforcement of rules and sanctions, and users' participation in crafting the rules were the major variables that strengthen collective action in forest fire management. Conversely, users' dependency on a daily wage and a lack of transparency were the variables that weaken collective action. In fire-prone forests such as the Siwalik, our results indicate that strengthening social capital and forming and enforcing forest fire management rules are important variables that encourage people to engage in collective action in fire management. PMID:25413128

  7. Streamlined Approach for Environmental Restoration Plan for Corrective Action Unit 484: Surface Debris, Waste Sites, and Burn Area, Tonopah Test Range, Nevada

    SciTech Connect

    Bechel Nevada

    2004-05-01

    This Streamlined Approach for Environmental Restoration plan details the activities necessary to close Corrective Action Unit (CAU) 484: Surface Debris, Waste Sites, and Burn Area (Tonopah Test Range). CAU 484 consists of sites located at the Tonopah Test Range, Nevada, and is currently listed in Appendix III of the Federal Facility Agreement and Consent Order. CAU 484 consists of the following six Corrective Action Sites: (1) CAS RG-52-007-TAML, Davis Gun Penetrator Test; (2) CAS TA-52-001-TANL, NEDS Detonation Area; (3) CAS TA-52-004-TAAL, Metal Particle Dispersion Test; (4) CAS TA-52-005-TAAL, Joint Test Assembly DU Sites; (5) CAS TA-52-006-TAPL, Depleted Uranium Site; and (6) CAS TA-54-001-TANL, Containment Tank and Steel Structure

  8. CORRECTIVE ACTION PLAN FOR CORRECTIVE ACTION UNIT 536: AREA 3 RELEASE SITE, NEVADA TEST SITE, NEVADA

    SciTech Connect

    2005-09-01

    CAU 536 consists of CAS 03-44-02, Steam Jenny Discharge, located in Area 3 of the NTS. The site was characterized in 2004 according to the approved CAIP and the site characterization results are reported in the CAU 536 CADD. The purpose of this Corrective Action Plan (CAP) is to provide the detailed scope of work required to implement the recommended corrective actions as specified in the approved CAU 536 CADD.

  9. Experimental Semiotics: A New Approach For Studying Communication As A Form Of Joint Action

    PubMed Central

    Galantucci, Bruno

    2015-01-01

    In the last few years, researchers have begun to investigate the emergence of novel forms of human communication in the laboratory. I survey this growing line of research, which may be called experimental semiotics, from three distinct angles. First, I situate the new approach in its theoretical and historical context. Second, I review a sample of studies that exemplify experimental semiotics. Third, I present an empirical study that illustrates how the new approach can help us understand the sociocognitive underpinnings of human communication. The main conclusion of the paper will be that, by reproducing micro samples of historical processes in the laboratory, experimental semiotics offers new powerful tools for investigating human communication as a form of joint action. PMID:25164941

  10. Differentiation of teaching and learning mathematics: an action research study in tertiary education

    NASA Astrophysics Data System (ADS)

    Konstantinou-Katzi, Panagiota; Tsolaki, Eleni; Meletiou-Mavrotheris, Maria; Koutselini, Mary

    2013-04-01

    Diversity and differentiation within our classrooms, at all levels of education, is nowadays a fact. It has been one of the biggest challenges for educators to respond to the needs of all students in such a mixed-ability classroom. Teachers' inability to deal with students with different levels of readiness in a different way leads to school failure and all the negative outcomes that come with it. Differentiation of teaching and learning helps addressing this problem by respecting the different levels that exist in the classroom, and by responding to the needs of each learner. This article presents an action research study where a team of mathematics instructors and an expert in curriculum development developed and implemented a differentiated instruction learning environment in a first-year engineering calculus class at a university in Cyprus. This study provides evidence that differentiated instruction has a positive effect on student engagement and motivation and improves students' understanding of difficult calculus concepts.

  11. NTP technical report on the toxicology and carcinogenesis studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (CAS No. 1746-01-6) in female Harlan Sprague-Dawley rats (Gavage Studies).

    PubMed

    2006-04-01

    DIOXIN TOXIC EQUIVALENCY FACTOR EVALUATION OVERVIEW: Polyhalogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have the ability to bind to and activate the ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR). Structurally related compounds that bind to the AhR and exhibit biological actions similar to TCDD are commonly referred to as "dioxin-like compounds" (DLCs). Ambient human exposure to DLCs occurs through the ingestion of foods containing residues of DLCs that bioconcentrate through the food chain. Due to their lipophilicity and persistence, once internalized, they accumulate in body tissue, mainly adipose, resulting in chronic lifetime human exposure. Since human exposure to DLCs always involves a complex mixture, the toxic equivalency factor (TEF) methodology has been developed as a mathematical tool to assess the health risk posed by complex mixtures of these compounds. The TEF methodology is a relative potency scheme that ranks the dioxin-like activity of a compound relative to TCDD, which is the most potent congener. This allows for the estimation of the potential dioxin-like activity of a mixture of chemicals based on a common mechanism of action involving an initial binding of DLCs to the AhR. The toxic equivalency of DLCs was nominated for evaluation because of the widespread human exposure to DLCs and the lack of data on the adequacy of the TEF methodology for predicting relative potency for cancer risk. To address this, the National Toxicology Program conducted a series of 2-year bioassays in female Harlan Sprague-Dawley rats to evaluate the chronic toxicity and carcinogenicity of DLCs and structurally related polychlorinated biphenyls (PCBs) and mixtures of these compounds. TCDD is not manufactured commercially other than for scientific research purposes. The main sources of TCDD releases into the environment are from combustion and incineration; metal smelting, refining, and processing; chemical

  12. Repurposing the CRISPR-Cas9 system for targeted DNA methylation.

    PubMed

    Vojta, Aleksandar; Dobrinić, Paula; Tadić, Vanja; Bočkor, Luka; Korać, Petra; Julg, Boris; Klasić, Marija; Zoldoš, Vlatka

    2016-07-01

    Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co-expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression. PMID:26969735

  13. Repurposing the CRISPR-Cas9 system for targeted DNA methylation

    PubMed Central

    Vojta, Aleksandar; Dobrinić, Paula; Tadić, Vanja; Bočkor, Luka; Korać, Petra; Julg, Boris; Klasić, Marija; Zoldoš, Vlatka

    2016-01-01

    Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co–expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression. PMID:26969735

  14. Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists.

    PubMed

    Lander, Noelia; Chiurillo, Miguel A; Docampo, Roberto

    2016-09-01

    Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii. PMID:27315329

  15. One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system

    PubMed Central

    Wang, Xianlong; Cao, Chunwei; Huang, Jiaojiao; Yao, Jing; Hai, Tang; Zheng, Qiantao; Wang, Xiao; Zhang, Hongyong; Qin, Guosong; Cheng, Jinbo; Wang, Yanfang; Yuan, Zengqiang; Zhou, Qi; Wang, Hongmei; Zhao, Jianguo

    2016-01-01

    Pig shows multiple superior characteristics in anatomy, physiology, and genome that have made this species to be more suitable models for human diseases, especially for neurodegenerative diseases, because they have similar cerebral convolutions compared with human neocortex. Recently, CRISPR/Cas9 system shows enormous potential for engineering the pig genome. In this study, we expect to generate human Parkinson’s disease pig model using CRISPR/Cas9 system by simultaneously targeting three distinct genomic loci, parkin/DJ-1/PINK1, in Bama miniature pigs. By co-injection of Cas9 mRNA and multiplexing single guide RNAs (sgRNAs) targeting parkin, DJ-1, and PINK1 genes, respectively, into in vivo derived pronuclear embryos, we simultaneously targeted three distinct genomic loci. The gene modified piglets remain healthy and display normal behavior at the age of 10 months. In addition, despite the high number of sgRNAs were employed in the present study, our trio-based whole-genome sequencing analysis suggested that the incidence of off-target events is low. Our results demonstrate that the simplicity, efficiency, and power of the CRISPR/Cas9 system to allow for the modification of multiple genes in pigs and yield results of high medical value. PMID:26857844

  16. Do emotions or gender drive our actions? A study of motor distractibility.

    PubMed

    Ambron, Elisabetta; Rumiati, Raffaella I; Foroni, Francesco

    2016-01-01

    People's interaction with the social environment depends on the ability to attend social cues with human faces being a key vehicle of this information. This study explores whether directing the attention to gender or emotion of a face interferes with ongoing actions. In two experiments, participants reached for one of two possible targets by relying on one of two features of a face, namely, emotion (Experiment 1) or gender (Experiment 2) of a non-target stimulus (a task-relevant distractor). Participants' reaching movements deviated toward the task-relevant distractor in both experiments. However, when attending to the gender of the face the distractor effect was modulated by both gender (task-relevant feature) and emotion (task-irrelevant feature), with the largest movement deviation being observed toward angry male faces. Endogenous allocation of attention toward faces elicits a competing motor response to the ongoing action and the emotional content of the face contributes to this process at a more automatic and implicit level. PMID:26418348

  17. Eating tools in hand activate the brain systems for eating action: a transcranial magnetic stimulation study.

    PubMed

    Yamaguchi, Kaori; Nakamura, Kimihiro; Oga, Tatsuhide; Nakajima, Yasoichi

    2014-07-01

    There is increasing neuroimaging evidence suggesting that visually presented tools automatically activate the human sensorimotor system coding learned motor actions relevant to the visual stimuli. Such crossmodal activation may reflect a general functional property of the human motor memory and thus can be operating in other, non-limb effector organs, such as the orofacial system involved in eating. In the present study, we predicted that somatosensory signals produced by eating tools in hand covertly activate the neuromuscular systems involved in eating action. In Experiments 1 and 2, we measured motor evoked response (MEP) of the masseter muscle in normal humans to examine the possible impact of tools in hand (chopsticks and scissors) on the neuromuscular systems during the observation of food stimuli. We found that eating tools (chopsticks) enhanced the masseter MEPs more greatly than other tools (scissors) during the visual recognition of food, although this covert change in motor excitability was not detectable at the behavioral level. In Experiment 3, we further observed that chopsticks overall increased MEPs more greatly than scissors and this tool-driven increase of MEPs was greater when participants viewed food stimuli than when they viewed non-food stimuli. A joint analysis of the three experiments confirmed a significant impact of eating tools on the masseter MEPs during food recognition. Taken together, these results suggest that eating tools in hand exert a category-specific impact on the neuromuscular system for eating. PMID:24835403

  18. Numerical Studies of Dynamo Action in a Turbulent Shear Flow. I.

    NASA Astrophysics Data System (ADS)

    Singh, Nishant K.; Jingade, Naveen

    2015-06-01

    We perform numerical experiments to study the shear dynamo problem where we look for the growth of a large-scale magnetic field due to non-helical stirring at small scales in a background linear shear flow in previously unexplored parameter regimes. We demonstrate the large-scale dynamo action in the limit where the fluid Reynolds number (\\operatorname{Re}) is below unity while the magnetic Reynolds number (Rm) is above unity; the exponential growth rate scales linearly with shear, which is consistent with earlier numerical works. The limit of low \\operatorname{Re} is particularly interesting, as seeing the dynamo action in this limit would provide enough motivation for further theoretical investigations, which may focus attention on this analytically more tractable limit of \\operatorname{Re}\\lt 1 compared to the more formidable limit of \\operatorname{Re}\\gt 1. We also perform simulations in the regimes where (i) both (\\operatorname{Re}, Rm) < 1, and (ii) \\operatorname{Re}\\gt 1 and Rm\\lt 1, and compute all of the components of the turbulent transport coefficients ({{α }ij} and {{η }ij}) using the test-field method. A reasonably good agreement is observed between our results and the results of earlier analytical works in similar parameter regimes.

  19. Health activism and the logic of connective action. A case study of rare disease patient organisations

    PubMed Central

    Vicari, Stefania; Cappai, Franco

    2016-01-01

    ABSTRACT This exploratory work investigates the role of digital media in expanding health discourse practices in a way to transform traditional structures of agency in public health. By focusing on a sample of rare disease patient organisations as representative of contemporary health activism, this study investigates the role of digital communication in the development of (1) bottom-up sharing and co-production of health knowledge, (2) health public engagement dynamics and (3) health information pathways. Findings show that digital media affordances for patient organisations go beyond the provision of social support for patient communities; they ease one-way, two-way and crowdsourced processes of health knowledge sharing, exchange and co-production, provide personalised routes to health public engagement and bolster the emergence of varied pathways to health information where experiential knowledge and medical authority are equally valued. These forms of organisationally enabled connective action can help the surfacing of personal narratives that strengthen patient communities, the bottom-up production of health knowledge relevant to a wider public and the development of an informational and eventually cultural context that eases patients’ political action. PMID:27499676

  20. Collaborative agency to support integrated care for children, young people and families: an action research study

    PubMed Central

    Stuart, Kaz

    2014-01-01

    Abstract Introduction Collaboration was legislated in the delivery of integrated care in the early 2000s in the UK. This research explored how the reality of practice met the rhetoric of collaboration. Theory The paper is situated against a theoretical framework of structure, agency, identity and empowerment. Collectively and contextually these concepts inform the proposed model of ‘collaborative agency’ to sustain integrated care. The paper brings sociological theory on structure and agency to the dilemma of collaboration. Methods Participative action research was carried out in collaborative teams that aspired to achieve integrated care for children, young people and families between 2009 and 2013. It was a part time, PhD study in collaborative practice. Results The research established that people needed to be able to be jointly aware of their context, to make joint decisions, and jointly act in order to deliver integrated services, and proposes a model of collaborative agency derived from practitioner’s experiences and integrated action research and literature on agency. The model reflects the effects of a range of structures in shaping professional identity, empowerment, and agency in a dynamic. The author proposes that the collaborative agency model will support integrated care, although this is, as yet, an untested hypothesis. PMID:24868192

  1. Exploring the meaning of recovery-oriented care: an action-research study.

    PubMed

    Kidd, Susan; Kenny, Amanda; McKinstry, Carol

    2015-02-01

    The present study describes participants' perspectives of the meaning of recovery-oriented care in developing services for people with psychosocial disability associated with mental illness. Participants were involved in a 12-month cooperative inquiry action-research group from August 2012 to July 2013, with six consumers, four clinicians, and a carer. A major finding was the importance of the facilitation of dialogue that acknowledged the asymmetrical power differences between participants. Thematically-analysed data identified an overarching global theme: 'I want services to hear me'. The theme reflected a shared view that participation is important in service development. Actions included mapping the integration of consumer participation within a mental health service and developing workshops to support change. Addressing the asymmetrical power relationship inherent in traditional mental health design is important. Using participatory processes, structural discrimination is revealed, and tensions associated with clinical mental health services and psychiatric practice can be discussed. A partnership approach to service development enables the social determinants of health to be addressed more effectively, as well as supporting individual recovery. These approaches create the potential for genuine transformational change. Approaches that support coproduction and codesign have the potential to enable solutions. PMID:25394375

  2. La tuberculose cutanée: observation de six cas confirmés au CHU Souro SANOU (CHUSS) de Bobo-Dioulasso (Burkina Faso)

    PubMed Central

    Andonaba, Jean Baptiste; Barro-Traoré, Fatou; Yaméogo, Téné; Diallo, Boukary; Korsaga-Somé, Nina; Traoré, Adama

    2013-01-01

    La localisation cutanée de la maladie tuberculeuse demeure une forme rare et représente seulement 2,1% des localisations. L'objet de cette étude est de rapporter le profil épidémiologique, anatomoclinique et évolutif des cas de tuberculose ganglio-cutanée diagnostiqués dans un CHU au Burkina Faso. La fréquence de la tuberculose cutanée est très faible au CHUSS. Six cas ont été diagnostiqués entre 2004 et 2010, soit une fréquence de un cas par an. La durée d’évolution des cas allait de deux jusqu’à dix ans avant leur diagnostic. Les lésions observées étaient: trois scrofulodermes, trois gommes, une tuberculose testiculaire associée à un mal de Pott, un cas de polyadénopathies et des cicatrices atropho-rétractiles dans la plupart des cas. Sur le plan anatomopathologique, des granulomes tuberculoïdes ont été mis en évidence dans tous les cas avec une forte réaction tuberculinique à l'IDR. Sous antituberculeux pendant six mois, l’évolution a été bonne dans tous les cas mais au prix de séquelles cutanées cicatricielles inesthétiques. Son ampleur reste peut-être encore méconnue. Le renforcement du plateau technique du CHU et une bonne collaboration interdisciplinaire contribuerait à un meilleur diagnostic et prise en charge de cette affection. PMID:24648863

  3. Corrective Action Decision Document/Closure Report for Corrective Action Unit 367: Area 10 Sedan, Ess and Uncle Unit Craters Nevada National Security Site, Nevada, Revision 0

    SciTech Connect

    Patrick Matthews

    2011-06-01

    Corrective Action Unit 367 comprises four corrective action sites (CASs): • 10-09-03, Mud Pit • 10-45-01, U-10h Crater (Sedan) • 10-45-02, Ess Crater Site • 10-45-03, Uncle Crater Site The purpose of this Corrective Action Decision Document/Closure Report is to provide justification and documentation of the corrective actions and site closure activities implemented at CAU 367. A corrective action of closure in place with use restrictions was completed at each of the three crater CASs (10-45-01, 10-45-02, and 10-45-03); corrective actions were not required at CAS 10-09-03. In addition, a limited soil removal corrective action was conducted at the location of a potential source material release. Based on completion of these correction actions, no additional corrective action is required at CAU 367, and site closure is considered complete. Corrective action investigation (CAI) activities were performed from February 2010 through March 2011, as set forth in the Corrective Action Investigation Plan for Corrective Action Unit 367: Area 10 Sedan, Ess and Uncle Unit Craters, Nevada Test Site, Nevada. The approach for the CAI was divided into two facets: investigation of the primary release of radionuclides, and investigation of non-test or other releases (e.g., migration in washes and potential source material). Based on the proximity of the Uncle, Ess, and Sedan craters, the impact of the Sedan test on the fallout deposited from the two earlier tests, and aerial radiological surveys, the CAU 367 investigation was designed to study the releases from the three crater CASs as one combined release (primary release). Corrective Action Site 10-09-03, Mud Pit, consists of two mud pits identified at CAU 367. The mud pits are considered non-test releases or other releases and were investigated independent of the three crater CASs. The purpose of the CAI was to fulfill data needs as defined during the data quality objective (DQO) process. The CAU 367 dataset of

  4. CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells.

    PubMed

    Ryan, Owen W; Poddar, Snigdha; Cate, Jamie H D

    2016-01-01

    This protocol describes a method for CRISPR-Cas9-mediated genome editing that results in scarless and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule that is used to repair the chromosomal DNA damage by homology-directed repair. For target specificity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA sequence within the sgRNA cassette. This CRISPR-Cas9 protocol includes methods for (1) cloning the unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides, and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial polyploid isolates. Therefore, this CRISPR-Cas9-based DNA integration protocol is achievable by virtually any yeast genetics and molecular biology laboratory. PMID:27250940

  5. Semiotic and Discursive Variables in CAS-Based Didactical Engineering.

    ERIC Educational Resources Information Center

    Winslow, Carl

    2003-01-01

    Presents a semiotic analysis of the potential of computer algebra systems (CAS) for enabling mathematical activity on a conceptual level higher than usual. Illustrates examples of theoretical points from a development project in the context of a first year university course in calculus. Discusses how CAS may be used in a didactical analysis and in…

  6. Transformation of OODT CAS to Perform Larger Tasks

    NASA Technical Reports Server (NTRS)

    Mattmann, Chris; Freeborn, Dana; Crichton, Daniel; Hughes, John; Ramirez, Paul; Hardman, Sean; Woollard, David; Kelly, Sean

    2008-01-01

    A computer program denoted OODT CAS has been transformed to enable performance of larger tasks that involve greatly increased data volumes and increasingly intensive processing of data on heterogeneous, geographically dispersed computers. Prior to the transformation, OODT CAS (also alternatively denoted, simply, 'CAS') [wherein 'OODT' signifies 'Object-Oriented Data Technology' and 'CAS' signifies 'Catalog and Archive Service'] was a proven software component used to manage scientific data from spaceflight missions. In the transformation, CAS was split into two separate components representing its canonical capabilities: file management and workflow management. In addition, CAS was augmented by addition of a resource-management component. This third component enables CAS to manage heterogeneous computing by use of diverse resources, including high-performance clusters of computers, commodity computing hardware, and grid computing infrastructures. CAS is now more easily maintainable, evolvable, and reusable. These components can be used separately or, taking advantage of synergies, can be used together. Other elements of the transformation included addition of a separate Web presentation layer that supports distribution of data products via Really Simple Syndication (RSS) feeds, and provision for full Resource Description Framework (RDF) exports of metadata.

  7. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  8. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  9. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  10. Teaching Undergraduate Mathematics Using CAS Technology: Issues and Prospects

    ERIC Educational Resources Information Center

    Tobin, Patrick C.; Weiss, Vida

    2016-01-01

    The use of handheld CAS technology in undergraduate mathematics courses in Australia is paradoxically shrinking under sustained disapproval or disdain from the professional mathematics community. Mathematics education specialists argue with their mathematics colleagues over a range of issues in course development and this use of CAS or even…

  11. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  12. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  13. 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. PMID:27340770

  14. Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae.

    PubMed

    Generoso, Wesley Cardoso; Gottardi, Manuela; Oreb, Mislav; Boles, Eckhard

    2016-08-01

    CRISPR-Cas has become a powerful technique for genetic engineering of yeast. Here, we present an improved version by using only one single plasmid expressing Cas9 and one or two guide-RNAs. A high gene deletion efficiency was achieved even with simultaneous recombination cloning of the plasmid and deletion in industrial strains. PMID:27327211

  15. Recent Progress in CRISPR/Cas9 Technology.

    PubMed

    Mei, Yue; Wang, Yan; Chen, Huiqian; Sun, Zhong Sheng; Ju, Xing-Da

    2016-02-20

    The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, a simple and efficient tool for genome editing, has experienced rapid progress in its technology and applicability in the past two years. Here, we review the recent advances in CRISPR/Cas9 technology and the ways that have been adopted to expand our capacity for precise genome manipulation. First, we introduce the mechanism of CRISPR/Cas9, including its biochemical and structural implications. Second, we highlight the latest improvements in the CRISPR/Cas9 system, especially Cas9 protein modifications for customization. Third, we review its current applications, in which the versatile CRISPR/Cas9 system was employed to edit the genome, epigenome, or RNA of various organisms. Although CRISPR/Cas9 allows convenient genome editing accompanied by many benefits, we should not ignore the significant ethical and biosafety concerns that it raises. Finally, we discuss the prospective applications and challenges of several promising techniques adapted from CRISPR/Cas9. PMID:26924689

  16. Gender differences in non-standard mapping tasks: A kinematic study using pantomimed reach-to-grasp actions.

    PubMed

    Copley-Mills, Freya; Connolly, Jason D; Cavina-Pratesi, Cristiana

    2016-09-01

    Comparison between real and pantomimed actions is used in neuroscience to dissociate stimulus-driven (real) as compared to internally driven (pantomimed) visuomotor transformations, with the goal of testing models of vision (Milner & Goodale, 1995) and diagnosing neuropsychological deficits (apraxia syndrome). Real actions refer to an overt movement directed toward a visible target whereas pantomimed actions refer to an overt movement directed either toward an object that is no longer available. Although similar, real and pantomimed actions differ in their kinematic parameters and in their neural substrates. Pantomimed-reach-to-grasp-actions show reduced reaching velocities, higher wrist movements, and reduced grip apertures. In addition, seminal neuropsychological studies and recent neuroimaging findings confirmed that real and pantomimed actions are underpinned by separate brain networks. Although previous literature suggests differences in the praxis system between males and females, no research to date has investigated whether or not gender differences exist in the context of real versus pantomimed reach-to-grasp actions. We asked ten male and ten female participants to perform real and pantomimed reach-to-grasp actions toward objects of different sizes, either with or without visual feedback. During pantomimed actions participants were required to pick up an imaginary object slightly offset relative to the location of the real one (which was in turn the target of the real reach-to-grasp actions). Results demonstrate a significant difference between the kinematic parameters recorded in male and female participants performing pantomimed, but not real reach-to-grasp tasks, depending on the availability of visual feedback. With no feedback both males and females showed smaller grip aperture, slower movement velocity and lower reach height. Crucially, these same differences were abolished when visual feedback was available in male, but not in female participants

  17. Comparative human bioavailability study of macrocrystalline nitrofurantoin and two prolonged-action hydroxymethylnitrofurantoin preparations.

    PubMed

    Guelen, P J; Boerema, J B; Vree, T B

    1988-12-01

    This single-blind crossover study compared the human bioavailability of macrocrystalline nitrofurantoin (Furadantine MC) and two prolonged-action hydroxymethylnitrofurantoin formulations (Urfadyn PL, bid, and Uridurine, tid), based on plasma nitrofurantoin concentrations and urinary nitrofurantoin excretion. The drugs were administered to 16 healthy females for a single day according to the recommended daily dosages. For comparison, Furadantine MC was administered both at the qid dosage recommended by the manufacturer and at tid dosage. Pharmacokinetic parameters determined were maximum plasma concentration after first dose, minimum plasma concentration after first dose, area under the plasma concentration-time curve (AUC), cumulative renal excretion over 30 hours (ARE), overall renal clearance, total body clearance, and bioavailability relative to Furadantine MC qid, based on plasma AUC (F) and ARE (Fren). F for Furadantine MC 100 mg tid was 108 +/- 25 percent (mean +/- SD); for Uridurine 100 mg tid and Urfadyn PL 100 mg bid, F equalled 86 +/- 33 percent and 53 +/- 20 percent (p less than 0.05), respectively. A similar relationship was observed between Fren for Furadantine MC 100 mg qid and the respective Fren of Furadantine MC 100 mg tid, Uridurine 100 mg tid, and Urfadyn PL 100 mg bid. No significant difference was found between the respective F and Fren of each of the drugs studied. Although bioavailability was comparable for Furadantine MC tid and qid, the single-day design of these studies precludes inferring that these dosage schedules are therapeutically equivalent. However, the significantly lower relative bioavailabilities for the prolonged-action hydroxymethylnitrofurantoin formulations suggest that Urfadyn PL 100 mg bid and Uridurine 100 mg tid are not pharmacokinetically equivalent to Furadantine MC. PMID:3243175

  18. Cas9-mediated targeting of viral RNA in eukaryotic cells

    PubMed Central

    Price, Aryn A.; Sampson, Timothy R.; Ratner, Hannah K.; Grakoui, Arash; Weiss, David S.

    2015-01-01

    Clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) systems are prokaryotic RNA-directed endonuclease machineries that act as an adaptive immune system against foreign genetic elements. Using small CRISPR RNAs that provide specificity, Cas proteins recognize and degrade nucleic acids. Our previous work demonstrated that the Cas9 endonuclease from Francisella novicida (FnCas9) is capable of targeting endogenous bacterial RNA. Here, we show that FnCas9 can be directed by an engineered RNA-targeting guide RNA to target and inhibit a human +ssRNA virus, hepatitis C virus, within eukaryotic cells. This work reveals a versatile and portable RNA-targeting system that can effectively function in eukaryotic cells and be programmed as an antiviral defense. PMID:25918406

  19. Exploiting the CRISPR/Cas9 System for Targeted Genome Mutagenesis in Petunia

    PubMed Central

    Zhang, Bin; Yang, Xia; Yang, Chunping; Li, Mingyang; Guo, Yulong

    2016-01-01

    Recently, CRISPR/Cas9 technology has emerged as a powerful approach for targeted genome modification in eukaryotic organisms from yeast to human cell lines. Its successful application in several plant species promises enormous potential for basic and applied plant research. However, extensive studies are still needed to assess this system in other important plant species, to broaden its fields of application and to improve methods. Here we showed that the CRISPR/Cas9 system is efficient in petunia (Petunia hybrid), an important ornamental plant and a model for comparative research. When PDS was used as target gene, transgenic shoot lines with albino phenotype accounted for 55.6%–87.5% of the total regenerated T0 Basta-resistant lines. A homozygous deletion close to 1 kb in length can be readily generated and identified in the first generation. A sequential transformation strategy—introducing Cas9 and sgRNA expression cassettes sequentially into petunia—can be used to make targeted mutations with short indels or chromosomal fragment deletions. Our results present a new plant species amenable to CRIPR/Cas9 technology and provide an alternative procedure for its exploitation. PMID:26837606

  20. CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.).

    PubMed

    Ren, Chong; Liu, Xianju; Zhang, Zhan; Wang, Yi; Duan, Wei; Li, Shaohua; Liang, Zhenchang

    2016-01-01

    The type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has been successfully applied to edit target genes in multiple plant species. However, it remains unknown whether this system can be used for genome editing in grape. In this study, we described genome editing and targeted gene mutation in 'Chardonnay' suspension cells and plants via the CRISPR/Cas9 system. Two single guide RNAs (sgRNAs) were designed to target distinct sites of the L-idonate dehydrogenase gene (IdnDH). CEL I endonuclease assay and sequencing results revealed the expected indel mutations at the target site, and a mutation frequency of 100% was observed in the transgenic cell mass (CM) as well as corresponding regenerated plants with expression of sgRNA1/Cas9. The majority of the detected mutations in transgenic CM were 1-bp insertions, followed by 1- to 3-nucleotide deletions. Off-target activities were also evaluated by sequencing the potential off-target sites, and no obvious off-target events were detected. Our results demonstrated that the CRISPR/Cas9 system is an efficient and specific tool for precise genome editing in grape. PMID:27576893