Science.gov

Sample records for activator-like effectors tales

  1. [Transcription activator-like effectors(TALEs)based genome engineering].

    PubMed

    Zhao, Mei-Wei; Duan, Cheng-Li; Liu, Jiang

    2013-10-01

    Systematic reverse-engineering of functional genome architecture requires precise modifications of gene sequences and transcription levels. The development and application of transcription activator-like effectors(TALEs) has created a wealth of genome engineering possibilities. TALEs are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas species. The DNA-binding domain of each TALE typically consists of tandem 34-amino acid repeat modules rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Such "genome engineering" has now been established in human cells and a number of model organisms, thus opening the door to better understanding gene function in model organisms, improving traits in crop plants and treating human genetic disorders.

  2. Overcoming transcription activator-like effector (TALE) DNA binding domain sensitivity to cytosine methylation.

    PubMed

    Valton, Julien; Dupuy, Aurélie; Daboussi, Fayza; Thomas, Séverine; Maréchal, Alan; Macmaster, Rachel; Melliand, Kevin; Juillerat, Alexandre; Duchateau, Philippe

    2012-11-09

    Within the past 2 years, transcription activator-like effector (TALE) DNA binding domains have emerged as the new generation of engineerable platform for production of custom DNA binding domains. However, their recently described sensitivity to cytosine methylation represents a major bottleneck for genome engineering applications. Using a combination of biochemical, structural, and cellular approaches, we were able to identify the molecular basis of such sensitivity and propose a simple, drug-free, and universal method to overcome it.

  3. Direct observation of transcription activator-like effector (TALE) protein dynamics

    NASA Astrophysics Data System (ADS)

    Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M.

    2014-03-01

    In this work, we describe a single molecule assay to probe the site-search dynamics of transcription activator-like effector (TALE) proteins along DNA. In modern genetics, the ability to selectively edit the human genome is an unprecedented development, driven by recent advances in targeted nuclease proteins. Specific gene editing can be accomplished using TALE proteins, which are programmable DNA-binding proteins that can be fused to a nuclease domain. In this way, TALENs are a leading technology that has shown great success in the genomic editing of pluripotent stem cells. A major hurdle facing clinical implementation, however, is the potential for deleterious off-target binding events. For these reasons, a molecular-level understanding of TALE binding and target sequence search on DNA is essential. To this end, we developed a single-molecule fluorescence imaging assay that provides a first-of-its-kind view of the 1-D diffusion of TALE proteins along stretched DNA. Taken together with co-crystal structures of DNA-bound TALEs, our results suggest a rotationally-coupled, major groove tracking model for diffusion. We further report diffusion constants for TALE proteins as a function of salt concentration, consistent with previously described models of 1-D protein diffusion.

  4. Locked and proteolysis-based transcription activator-like effector (TALE) regulation

    PubMed Central

    Lonzarić, Jan; Lebar, Tina; Majerle, Andreja; Manček-Keber, Mateja; Jerala, Roman

    2016-01-01

    Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE. PMID:26748097

  5. The last half-repeat of transcription activator-like effector (TALE) is dispensable and thereby TALE-based technology can be simplified.

    PubMed

    Zheng, Chong-Ke; Wang, Chun-Lian; Zhang, Xiao-Ping; Wang, Fu-Jun; Qin, Teng-Fei; Zhao, Kai-Jun

    2014-09-01

    To activate the expression of host genes that contribute to pathogen growth, pathogenic Xanthomonas bacteria inject their transcription activator-like effectors (TALEs) into plant cells and the TALEs bind to target gene promoters by the central repeat region consisting of near-perfect 34-amino-acid repeats (34-aa repeats). Based on the recognition codes between the 34-aa repeats and the targeted nucleotides, TALE-based technologies, such as designer TALEs (dTALEs) and TALE nucleases (TALENs), have been developed. Amazingly, every natural TALE invariantly has a truncated last half-repeat (LHR) at the end of the 34-aa repeats. Consequently, all the reported dTALEs and TALENs also harbour their LHRs. Here, we show that the LHRs in dTALEs are dispensable for the function of gene activation by both transient expression assays in Nicotiana benthamiana and gene-specific targeting in the rice genome, indicating that TALEs might originate from a single progenitor. In the light of this finding, we demonstrate that dTALEs can be constructed through two simple steps. Moreover, the activation strengths of dTALEs lacking the LHR are comparable with those of dTALEs harbouring the LHR. Our results provide new insights into the origin of natural TALEs, and will facilitate the simplification of the design and assembly of TALE-based tools, such as dTALEs and TALENs, in the near future.

  6. Transcription activator like effector (TALE)-directed piggyBac transposition in human cells.

    PubMed

    Owens, Jesse B; Mauro, Damiano; Stoytchev, Ilko; Bhakta, Mital S; Kim, Moon-Soo; Segal, David J; Moisyadi, Stefan

    2013-10-01

    Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations.

  7. Site-specific gene targeting using transcription activator-like effector (TALE)-based nuclease in Brassica oleracea.

    PubMed

    Sun, Zijian; Li, Nianzu; Huang, Guodong; Xu, Junqiang; Pan, Yu; Wang, Zhimin; Tang, Qinglin; Song, Ming; Wang, Xiaojia

    2013-11-01

    Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors (TALEs) contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases (TALENs), constructed using a method called "unit assembly", specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non-homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.

  8. De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks.

    PubMed

    Mahfouz, Magdy M; Li, Lixin; Shamimuzzaman, Md; Wibowo, Anjar; Fang, Xiaoyun; Zhu, Jian-Kang

    2011-02-08

    Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.

  9. A transcription activator-like effector toolbox for genome engineering.

    PubMed

    Sanjana, Neville E; Cong, Le; Zhou, Yang; Cunniff, Margaret M; Feng, Guoping; Zhang, Feng

    2012-01-05

    Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas sp. The DNA-binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within 1 week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using quantitative reverse-transcription PCR and Surveyor nuclease, respectively. The TALE toolbox described here will enable a broad range of biological applications.

  10. Transcription activator-like effector nucleases (TALENs): a highly efficient and versatile tool for genome editing.

    PubMed

    Sun, Ning; Zhao, Huimin

    2013-07-01

    Transcription activator-like effector (TALE) nucleases (TALENs) have recently emerged as a revolutionary genome editing tool in many different organisms and cell types. The site-specific chromosomal double-strand breaks introduced by TALENs significantly increase the efficiency of genomic modification. The modular nature of the TALE central repeat domains enables researchers to tailor DNA recognition specificity with ease and target essentially any desired DNA sequence. Here, we comprehensively review the development of TALEN technology in terms of scaffold optimization, DNA recognition, and repeat array assembly. In addition, we provide some perspectives on the future development of this technology.

  11. Rapid and efficient assembly of transcription activator-like effector genes by USER cloning.

    PubMed

    Wang, Song; Li, Wei; Wang, Shuo; Hu, Baoyang

    2014-06-20

    Transcription activator-like effectors (TALEs) that were related to bacteria immune system have lately been employed in a promising approach of precise gene targeting. Because of the repetitive characteristics of TALEs, existing TALE assembly methods are either very complicated, time-consuming, or too tricky to be handled in common labs. Here, we reported a rapid, efficient and easy method for TALE assembly. This method takes advantage of uracil-specific excision reagent (USER), an enzyme that can cleave DNA constructs and create long, unique single-strand DNA overhangs. Upon USER treatment, the overhangs on each individual TALE repeat unit can be rejoined hierarchically to form pentamers in a ligation-independent manner. Eventually, three pentamers are assembled into a full TALE construct by Golden Gate cloning. TALE nucleases (TALENs) generated with this method exhibit high genome-editing activity in human cells such as HEK293FT cells. Using this method, we have successfully synthesized three TALEN pairs targeting endogenous Tet1 locus, and proved that all can specifically target Tet1 gene, though in various degree. Comparing to other methods of TALEN assembly, this one is much less labor intensive and fairly faster, and positive clones can be obtained at high efficiency within only two days. We thus contribute to an easier approach for effective TALENs synthesis, which may highly facilitate the wide application of TALEN technology in genome editing, especially for human cells that require precise targeting.

  12. Exploring the transcription activator-like effectors scaffold versatility to expand the toolbox of designer nucleases

    PubMed Central

    2014-01-01

    Background The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases. Results In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold. In contrast to the existing Tail to Tail (TtT) and head to Head (HtH) nuclease architectures based on the symmetrical association of two TALE DNA binding domains fused to the C-terminal (TtT) or N-terminal (HtH) end of FokI, this novel architecture consists of the asymmetrical association of two different engineered TALE DNA binding domains fused to the N- and C-terminal ends of FokI (TALE::FokI and FokI::TALE scaffolds respectively). The characterization of this novel Tail to Head (TtH) architecture in yeast enabled us to demonstrate its nuclease activity and define its optimal target configuration. We further showed that this architecture was able to promote substantial level of targeted mutagenesis at three endogenous loci present in two different mammalian cell lines. Conclusion Our results demonstrated that this novel functional TtH architecture which requires binding to only one DNA strand of a given endogenous locus has the potential to extend the targeting possibility of FokI-based TALE nucleases. PMID:24997498

  13. Comparing zinc finger nucleases and transcription activator-like effector nucleases for gene targeting in Drosophila.

    PubMed

    Beumer, Kelly J; Trautman, Jonathan K; Christian, Michelle; Dahlem, Timothy J; Lake, Cathleen M; Hawley, R Scott; Grunwald, David J; Voytas, Daniel F; Carroll, Dana

    2013-10-03

    Zinc-finger nucleases have proven to be successful as reagents for targeted genome manipulation in Drosophila melanogaster and many other organisms. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of DNA recognition by zinc fingers. Transcription activator-like effector (TALE) DNA-binding domains depend on a simple, one-module-to-one-base-pair recognition code, and they have been very productively incorporated into nucleases (TALENs) for genome engineering. In this report we describe the design of TALENs for a number of different genes in Drosophila, and we explore several parameters of TALEN design. The rate of success with TALENs was substantially greater than for zinc-finger nucleases , and the frequency of mutagenesis was comparable. Knockout mutations were isolated in several genes in which such alleles were not previously available. TALENs are an effective tool for targeted genome manipulation in Drosophila.

  14. FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science.

    PubMed

    Ma, Alvin C; McNulty, Melissa S; Poshusta, Tanya L; Campbell, Jarryd M; Martínez-Gálvez, Gabriel; Argue, David P; Lee, Han B; Urban, Mark D; Bullard, Cassandra E; Blackburn, Patrick R; Man, Toni K; Clark, Karl J; Ekker, Stephen C

    2016-06-01

    Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multitube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process, (3) requires only commonly used basic molecular biology reagents, and (4) is cost-effective. More than 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish, with each showing high somatic and germline activity. Furthermore, this assembly system is flexible and is compatible with standard molecular biology laboratory tools, but can be scaled with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid-handling robot to rapidly and accurately assemble TALEs using the FusX TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications.

  15. FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science

    PubMed Central

    Ma, Alvin C.; McNulty, Melissa S.; Poshusta, Tanya L.; Campbell, Jarryd M.; Martínez-Gálvez, Gabriel; Argue, David P.; Lee, Han B.; Urban, Mark D.; Bullard, Cassandra E.; Blackburn, Patrick R.; Man, Toni K.; Clark, Karl J.; Ekker, Stephen C.

    2016-01-01

    Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multitube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process, (3) requires only commonly used basic molecular biology reagents, and (4) is cost-effective. More than 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish, with each showing high somatic and germline activity. Furthermore, this assembly system is flexible and is compatible with standard molecular biology laboratory tools, but can be scaled with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid-handling robot to rapidly and accurately assemble TALEs using the FusX TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications. PMID:26854857

  16. Allele-Specific Reduction of the Mutant Huntingtin Allele Using Transcription Activator-Like Effectors in Human Huntington's Disease Fibroblasts.

    PubMed

    Fink, Kyle D; Deng, Peter; Gutierrez, Josh; Anderson, Joseph S; Torrest, Audrey; Komarla, Anvita; Kalomoiris, Stefanos; Cary, Whitney; Anderson, Johnathon D; Gruenloh, William; Duffy, Alexandra; Tempkin, Teresa; Annett, Geralyn; Wheelock, Vicki; Segal, David J; Nolta, Jan A

    2016-01-01

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of CAG repeats. Although pathogenesis has been attributed to this polyglutamine expansion, the underlying mechanisms through which the huntingtin protein functions have yet to be elucidated. It has been suggested that postnatal reduction of mutant huntingtin through protein interference or conditional gene knockout could prove to be an effective therapy for patients suffering from HD. For allele-specific targeting, transcription activator-like effectors (TALE) were designed to target single-nucleotide polymorphisms (SNP) in the mutant allele and packaged into a vector backbone containing KRAB to promote transcriptional repression of the disease-associated allele. Additional TALEs were packaged into a vector backbone containing heterodimeric FokI and were designed to be used as nucleases (TALEN) to cause a CAG-collapse in the mutant allele. Human HD fibroblasts were treated with each TALE-SNP or TALEN. Allele-expression was measured using a SNP-genotyping assay and mutant protein aggregation was quantified with Western blots for anti-ubiquitin. The TALE-SNP and TALEN significantly reduced mutant allele expression (p < 0.05) when compared to control transfections while not affecting expression of the nondisease allele. This study demonstrates the potential of allele-specific gene modification using TALE proteins, and provides a foundation for targeted treatment for individuals suffering from Huntington's or other genetically linked diseases.

  17. A One-Step System for Convenient and Flexible Assembly of Transcription Activator-Like Effector Nucleases (TALENs).

    PubMed

    Zhao, Jinlong; Sun, Wenye; Liang, Jing; Jiang, Jing; Wu, Zhao

    2016-09-01

    Transcription activator-like effector nucleases (TALENs) are powerful tools for targeted genome editing in diverse cell types and organisms. However, the highly identical TALE repeat sequences make it challenging to assemble TALEs using conventional cloning approaches, and multiple repeats in one plasmid are easily catalyzed for homologous recombination in bacteria. Although the methods for TALE assembly are constantly improving, these methods are not convenient because of laborious assembly steps or large module libraries, limiting their broad utility. To overcome the barrier of multiple assembly steps, we report a one-step system for the convenient and flexible assembly of a 180 TALE module library. This study is the first demonstration to ligate 9 mono-/dimer modules and one circular TALEN backbone vector in a one step process, generating 9.5 to 18.5 repeat sequences with an overall assembly rate higher than 50%. This system makes TALEN assembly much simpler than the conventional cloning of two DNA fragments because this strategy combines digestion and ligation into one step using circular vectors and different modules to avoid gel extraction. Therefore, this system provides a convenient tool for the application of TALEN-mediated genome editing in scientific studies and clinical trials.

  18. Genetic diversity of transcriptional activator-like effector genes in Chinese isolates of Xanthomonas oryzae pv. oryzicola.

    PubMed

    Ji, Zhi-Yuan; Zakria, Muhammad; Zou, Li-Fang; Xiong, Li; Li, Zheng; Ji, Guang-Hai; Chen, Gong-You

    2014-07-01

    Xanthomonas oryzae pv. oryzicola causes bacterial leaf streak (BLS), a devastating disease of rice in Asia countries. X. oryzae pv. oryzicola utilizes repertoires of transcriptional activator-like effectors (TALEs) to manipulate host resistance or susceptibility; thus, TALEs can determine the outcome of BLS. In this report, we studied genetic diversity in putative tale genes of 65 X. oryzae pv. oryzicola strains that originated from nine provinces of southern China. Genomic DNAs from the 65 strains were digested with BamHI and hybridized with an internal fragment of avrXa3, a tale gene originating from the related pathogen, X. oryzae pv. oryzae, which causes bacterial leaf blight (BLB). Southern blot analysis indicated that the strains contained a variable number (9 to 22) of avrXa3-hybridizing fragments (e.g., putative tale genes). Based on the number and size of hybridizing bands, strains were classified into 14 genotypes (designated 1 to 14), and genotypes 3 and 10 represented 29.23 and 24.64% of the total, respectively. A high molecular weight BamHI fragment (HMWB; ≈6.0 kb) was present in 12 of the 14 genotypes, and sequence analysis of the HMWB revealed the presence of a C-terminally truncated tale, an insertion element related to IS1403, and genes encoding phosphoglycerate mutase and endonuclease V. Primers were developed from the 6.0-kb HMWB fragment and showed potential in genotyping X. oryzae pv. oryzicola strains by polymerase chain reaction. Virulence of X. oryzae pv. oryzicola strains was assessed on 23 rice cultivars containing different resistance genes for BLB. The X. oryzae pv. oryzicola strains could be grouped into 14 pathotypes (I to XIV), and the grouping of strains was almost identical to the categories determined by genotypic analysis. In general, strains containing higher numbers of putative tale genes were more virulent on rice than strains containing fewer tales. The results also indicate that there are no gene-for-gene relationships

  19. TALEs from a spring--superelasticity of Tal effector protein structures.

    PubMed

    Flechsig, Holger

    2014-01-01

    Transcription activator-like effectors (TALEs) are DNA-related proteins that recognise and bind specific target sequences to manipulate gene expression. Recently determined crystal structures show that their common architecture reveals a superhelical overall structure that may undergo drastic conformational changes. To establish a link between structure and dynamics in TALE proteins we have employed coarse-grained elastic-network modelling of currently available structural data and implemented a force-probe setup that allowed us to investigate their mechanical behaviour in computer experiments. Based on the measured force-extension curves we conclude that TALEs exhibit superelastic dynamical properties allowing for large-scale global conformational changes along their helical axis, which represents the soft direction in such proteins. For moderate external forcing the TALE models behave like linear springs, obeying Hooke's law, and the investigated structures can be characterised and compared by a corresponding spring constant. We show that conformational flexibility underlying the large-scale motions is not homogeneously distributed over the TALE structure, but instead soft spot residues around which strain is accumulated and which turn out to represent key agents in the transmission of conformational motions are identified. They correspond to the RVD loop residues that have been experimentally determined to play an eminent role in the binding process of target DNA.

  20. Transcription activator-like effector-mediated regulation of gene expression based on the inducible packaging and delivery via designed extracellular vesicles.

    PubMed

    Lainšček, Duško; Lebar, Tina; Jerala, Roman

    2017-02-26

    Transcription activator-like effector (TALE) proteins present a powerful tool for genome editing and engineering, enabling introduction of site-specific mutations, gene knockouts or regulation of the transcription levels of selected genes. TALE nucleases or TALE-based transcription regulators are introduced into mammalian cells mainly via delivery of the coding genes. Here we report an extracellular vesicle-mediated delivery of TALE transcription regulators and their ability to upregulate the reporter gene in target cells. Designed transcriptional activator TALE-VP16 fused to the appropriate dimerization domain was enriched as a cargo protein within extracellular vesicles produced by mammalian HEK293 cells stimulated by Ca-ionophore and using blue light- or rapamycin-inducible dimerization systems. Blue light illumination or rapamycin increased the amount of the TALE-VP16 activator in extracellular vesicles and their addition to the target cells resulted in an increased expression of the reporter gene upon addition of extracellular vesicles to the target cells. This technology therefore represents an efficient delivery for the TALE-based transcriptional regulators.

  1. Generation of knockout rabbits using transcription activator-like effector nucleases.

    PubMed

    Wang, Yu; Fan, Nana; Song, Jun; Zhong, Juan; Guo, Xiaogang; Tian, Weihua; Zhang, Quanjun; Cui, Fenggong; Li, Li; Newsome, Philip N; Frampton, Jon; Esteban, Miguel A; Lai, Liangxue

    2014-01-01

    Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

  2. Gene targeting in rats using transcription activator-like effector nucleases.

    PubMed

    Ménoret, Séverine; Tesson, Laurent; Rémy, Séverine; Usal, Claire; Thépenier, Virginie; Thinard, Reynald; Ouisse, Laure-Hélène; De Cian, Anne; Giovannangeli, Carine; Concordet, Jean-Paul; Anegon, Ignacio

    2014-08-15

    The rat is a model of choice to understanding gene function and modeling human diseases. Since recent years, successful engineering technologies using gene-specific nucleases have been developed to gene edit the genome of different species, including the rat. This development has become important for the creation of new rat animals models of human diseases, analyze the role of genes and express recombinant proteins. Transcription activator-like (TALE) nucleases are designed nucleases consist of a DNA binding domain fused to a nuclease domain capable of cleaving the targeted DNA. We describe a detailed protocol for generating knockout rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

  3. The application of transcription activator-like effector nucleases for genome editing in C. elegans.

    PubMed

    Yi, Peishan; Li, Wei; Ou, Guangshuo

    2014-08-01

    The nematode Caenorhabditis elegans has been a powerful model system for biomedical research in the past decades, however, the efficient genetic tools are still demanding for gene knockout, knock-in or conditional gene mutations. Transcription activator-like effector nucleases (TALENs) that comprise a sequence-specific DNA-binding domain fused to a FokI nuclease domain facilitate the targeted genome editing in various cell types or organisms. Here we summarize the recent progresses and protocols using TALENs in C. elegans that generate gene mutations and knock-ins in the germ line and the conditional gene knockout in somatic tissues.

  4. Genome Modification of Pluripotent Cells by Using Transcription Activator-Like Effector Nucleases (TALENs).

    PubMed

    Taheri-Ghahfarokhi, Amir; Malaver-Ortega, Luis F; Sumer, Huseyin

    2015-01-01

    Interest is increasing in transcription activator-like effector nucleases (TALENs) as a tool to introduce targeted double-strand breaks into the large genomes of human and animal cell lines. The produced DNA lesions stimulate DNA repair pathways, error-prone but dominant non-homologous end joining (NHEJ) and accurate but less occurring homology-directed repair (HDR), and as a result targeted genes can be modified. Here, we describe a modified Golden-Gate cloning method for generating TALENs and also details for targeting genes in mouse embryonic stem cells. The protocol described here can be used for modifying the genome of a broad range of pluripotent cell lines.

  5. Editing of the heavy chain gene of Bombyx mori using transcription activator like effector nucleases.

    PubMed

    Wang, Yujun; Nakagaki, Masao

    2014-07-18

    The silk gland of Bombyx mori represents an established in vivo system for producing recombinant proteins. However, low yields of recombinant proteins have limited the system's further development because endogenous silk proteins were present. Transcription activator-like effector nucleases (TALENs) tool which work in pairs to bind and cleave DNA at specific sites, have recently been shown to be effective for genome editing in various organisms, including silkworms. To improve the yield of recombinant proteins synthesized in the silkworm by eliminated competition with endogenous fibroin synthesis, the heavy chain (H-chain) gene was knocked out using transcription activator-like effector nucleases (TALENs). A pair of TALENs that targets the 1st exon in the H-chain gene was synthesized and microinjected into silkworm embryos; the injected silkworms were screened for H-chain gene knock out (H-KO) based on their sericin cocoon-making characteristics. Sequence analysis revealed that the H-chain of the mutation was successfully edited. The TALENs was very efficient in editing the genome DNA of silkworm. By being eliminated competition with the H-chain, the production of recombinant proteins would be expected to increase markedly if this H-KO system is used.

  6. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases.

    PubMed

    Zhao, Xinxia; Ni, Wei; Chen, Chuangfu; Sai, Wujiafu; Qiao, Jun; Sheng, Jingliang; Zhang, Hui; Li, Guozhong; Wang, Dawei; Hu, Shengwei

    2016-03-01

    Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

  7. Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs).

    PubMed

    Lei, Yong; Guo, Xiaogang; Liu, Yun; Cao, Yang; Deng, Yi; Chen, Xiongfeng; Cheng, Christopher H K; Dawid, Igor B; Chen, Yonglong; Zhao, Hui

    2012-10-23

    Transcription activator-like effector nucleases (TALENs) are an approach for directed gene disruption and have been proved to be effective in various animal models. Here, we report that TALENs can induce somatic mutations in Xenopus embryos with reliably high efficiency and that such mutations are heritable through germ-line transmission. We modified the Golden Gate method for TALEN assembly to make the product suitable for RNA transcription and microinjection into Xenopus embryos. Eight pairs of TALENs were constructed to target eight Xenopus genes, and all resulted in indel mutations with high efficiencies of up to 95.7% at the targeted loci. Furthermore, mutations induced by TALENs were highly efficiently passed through the germ line to F(1) frogs. Together with simple and reliable PCR-based approaches for detecting TALEN-induced mutations, our results indicate that TALENs are an effective tool for targeted gene editing/knockout in Xenopus.

  8. Generation of gene disruptions by transcription activator-like effector nucleases (TALENs) in Xenopus tropicalis embryos.

    PubMed

    Lei, Yong; Guo, Xiaogang; Deng, Yi; Chen, Yonglong; Zhao, Hui

    2013-05-10

    Transcription activator-like effector nucleases (TALENs) are novel engineered DNA nucleases, and have been proven to be effective for gene specific targeting in various species. Recently we reported gene disruptions in Xenopus embryos by using TALENs. Here we summarize the protocol that is used in our studies for gene disruption. This protocol covers selection of TALEN targeting sites, TALEN assembly with a modified Golden Gate method, and injection of TALEN mRNAs into Xenopus tropicalis embryos. We also provide details for detection of somatic and germ line transmitted mutations. And finally, we briefly describe establishment of knockout Xenopus lines. This protocol will facilitate broader applications of TALENs in studies of Xenopus biology.

  9. Engineering designer transcription activator-like effector nucleases (TALENs) by REAL or REAL-Fast assembly.

    PubMed

    Reyon, Deepak; Khayter, Cyd; Regan, Maureen R; Joung, J Keith; Sander, Jeffry D

    2012-10-01

    Engineered transcription activator-like effector nucleases (TALENs) are broadly useful tools for performing targeted genome editing in a wide variety of organisms and cell types including plants, zebrafish, C. elegans, rat, human somatic cells, and human pluripotent stem cells. Here we describe detailed protocols for the serial, hierarchical assembly of TALENs that require neither PCR nor specialized multi-fragment ligations and that can be implemented by any laboratory. These restriction enzyme and ligation (REAL)-based protocols can be practiced using plasmid libraries and user-friendly, Web-based software that both identifies target sites in sequences of interest and generates printable graphical guides that facilitate assembly of TALENs. With the described platform of reagents, protocols, and software, researchers can easily engineer multiple TALENs within 2 weeks using standard cloning techniques.

  10. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases

    PubMed Central

    Zhao, Xinxia; Ni, Wei; Chen, Chuangfu; Sai, Wujiafu; Qiao, Jun; Sheng, Jingliang; Zhang, Hui; Li, Guozhong; Wang, Dawei; Hu, Shengwei

    2016-01-01

    Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals. PMID:26950874

  11. Efficient targeted mutagenesis in medaka using custom-designed transcription activator-like effector nucleases.

    PubMed

    Ansai, Satoshi; Sakuma, Tetsushi; Yamamoto, Takashi; Ariga, Hiroyoshi; Uemura, Norihito; Takahashi, Ryosuke; Kinoshita, Masato

    2013-03-01

    Transcription activator-like effector nucleases (TALENs) have become powerful tools for targeted genome editing. Here we demonstrate efficient targeted mutagenesis in medaka (Oryzias latipes), which serves as an excellent vertebrate model for genetics and genomics. We designed and constructed a pair of TALENs targeting the medaka DJ-1 gene, a homolog of human DJ-1 (PARK7). These TALENs induced a number of insertions and deletions in the injected embryos with extremely high efficiency. This induction of mutations occurred in a dose-dependent manner. All screened G0 fish injected with the TALENs transmitted the TALEN-induced mutations to the next generation with high efficiency (44-100%). We also confirmed that these TALENs induced site-specific mutations because none of the mutations were found at potential off-target sites. In addition, the DJ-1 protein was lost in DJ-1(Δ7/Δ7) fish that carried a TALEN-induced frameshift mutation in both alleles. We also investigated the effect of the N- and C-terminal regions of the transcription activator-like (TAL) effector domain on the gene-disrupting activity of DJ1-TALENs and found that 287 amino acids at the N terminus and 63 amino acids at the C terminus of the TAL domain exhibited the highest disrupting activity in the injected embryos. Our results suggest that TALENs enable us to rapidly and efficiently establish knockout medaka strains. This is the first report of targeted mutagenesis in medaka using TALENs. The TALEN technology will expand the potential of medaka as a model system for genetics and genomics.

  12. Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases

    PubMed Central

    Ansai, Satoshi; Sakuma, Tetsushi; Yamamoto, Takashi; Ariga, Hiroyoshi; Uemura, Norihito; Takahashi, Ryosuke; Kinoshita, Masato

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) have become powerful tools for targeted genome editing. Here we demonstrate efficient targeted mutagenesis in medaka (Oryzias latipes), which serves as an excellent vertebrate model for genetics and genomics. We designed and constructed a pair of TALENs targeting the medaka DJ-1 gene, a homolog of human DJ-1 (PARK7). These TALENs induced a number of insertions and deletions in the injected embryos with extremely high efficiency. This induction of mutations occurred in a dose-dependent manner. All screened G0 fish injected with the TALENs transmitted the TALEN-induced mutations to the next generation with high efficiency (44–100%). We also confirmed that these TALENs induced site-specific mutations because none of the mutations were found at potential off-target sites. In addition, the DJ-1 protein was lost in DJ-1Δ7/Δ7 fish that carried a TALEN-induced frameshift mutation in both alleles. We also investigated the effect of the N- and C-terminal regions of the transcription activator-like (TAL) effector domain on the gene-disrupting activity of DJ1-TALENs and found that 287 amino acids at the N terminus and 63 amino acids at the C terminus of the TAL domain exhibited the highest disrupting activity in the injected embryos. Our results suggest that TALENs enable us to rapidly and efficiently establish knockout medaka strains. This is the first report of targeted mutagenesis in medaka using TALENs. The TALEN technology will expand the potential of medaka as a model system for genetics and genomics. PMID:23288935

  13. Rh D blood group conversion using transcription activator-like effector nucleases.

    PubMed

    Kim, Young-Hoon; Kim, Hyun O; Baek, Eun J; Kurita, Ryo; Cha, Hyuk-Jin; Nakamura, Yukio; Kim, Hyongbum

    2015-06-16

    Group O D-negative blood cells are universal donors in transfusion medicine and methods for converting other blood groups into this universal donor group have been researched. However, conversion of D-positive cells into D-negative is yet to be achieved, although conversion of group A or B cells into O cells has been reported. The Rh D blood group is determined by the RHD gene, which encodes a 12-transmembrane domain protein. Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs). After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained. Erythroid-lineage cells differentiated from these knockout erythroid progenitor cells do not agglutinate in the presence of anti-D reagents and do not express D antigen, as assessed using flow cytometry. Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine.

  14. Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs).

    PubMed

    Moore, Finola E; Reyon, Deepak; Sander, Jeffry D; Martinez, Sarah A; Blackburn, Jessica S; Khayter, Cyd; Ramirez, Cherie L; Joung, J Keith; Langenau, David M

    2012-01-01

    Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%-76.8% compared to 1.1%-3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

  15. Transcription Activator-Like Effector Nucleases (TALEN)-Mediated Targeted DNA Insertion in Potato Plants.

    PubMed

    Forsyth, Adrienne; Weeks, Troy; Richael, Craig; Duan, Hui

    2016-01-01

    Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. Specifically integrated transgenes are guaranteed to co-segregate, and expression level is more predictable, which makes downstream characterization and line selection more manageable. Because the site of DNA integration is known, the steps to deregulation of transgenic crops may be simplified. Here we describe a method that combines transcription activator-like effector nuclease (TALEN)-mediated induction of double strand breaks (DSBs) and non-autonomous marker selection to insert a transgene into a pre-selected, transcriptionally active region in the potato genome. In our experiment, TALEN was designed to create a DSB in the genome sequence following an endogenous constitutive promoter. A cytokinin vector was utilized for TALENs expression and prevention of stable integration of the nucleases. The donor vector contained a gene of interest cassette and a promoter-less plant-derived herbicide resistant gene positioned near the T-DNA left border which was used to select desired transgenic events. Our results indicated that TALEN induced T-DNA integration occurred with high frequency and resulting events have consistent expression of the gene of interest. Interestingly, it was found that, in most lines integration took place through one sided homology directed repair despite the minimal homologous sequence at the right border. An efficient transient assay for TALEN activity verification is also described.

  16. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases.

    PubMed

    Qiu, Zhongwei; Liu, Meizhen; Chen, Zhaohua; Shao, Yanjiao; Pan, Hongjie; Wei, Gaigai; Yu, Chao; Zhang, Long; Li, Xia; Wang, Ping; Fan, Heng-Yu; Du, Bing; Liu, Bin; Liu, Mingyao; Li, Dali

    2013-06-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.

  17. Comprehensive analysis of the specificity of transcription activator-like effector nucleases.

    PubMed

    Juillerat, Alexandre; Dubois, Gwendoline; Valton, Julien; Thomas, Séverine; Stella, Stefano; Maréchal, Alan; Langevin, Stéphanie; Benomari, Nassima; Bertonati, Claudia; Silva, George H; Daboussi, Fayza; Epinat, Jean-Charles; Montoya, Guillermo; Duclert, Aymeric; Duchateau, Philippe

    2014-04-01

    A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator-like effector nucleases (TALEN). The analysis of >15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only accommodate a relatively small number of position-dependent mismatches while maintaining a detectable activity at endogenous loci in vivo, demonstrating the high specificity of these molecular tools. We thus envision that the results we provide will allow for more deliberate choices of DNA binding arrays and/or DNA targets, extending our engineering capabilities.

  18. Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs).

    PubMed

    Kowalko, Johanna E; Ma, Li; Jeffery, William R

    2016-06-20

    Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs. Towards this end, much recent work has focused on identifying candidate genes for the evolution of traits in a variety of species. However, until recently it has been challenging to functionally validate interesting candidate genes. Recently developed tools for genetic engineering make it possible to manipulate specific genes in a wide range of organisms. Application of this technology in evolutionarily relevant organisms will allow for unprecedented insight into the role of candidate genes in evolution. Astyanax mexicanus (A. mexicanus) is a species of fish with both surface-dwelling and cave-dwelling forms. Multiple independent lines of cave-dwelling forms have evolved from ancestral surface fish, which are interfertile with one another and with surface fish, allowing elucidation of the genetic basis of cave traits. A. mexicanus has been used for a number of evolutionary studies, including linkage analysis to identify candidate genes responsible for a number of traits. Thus, A. mexicanus is an ideal system for the application of genome editing to test the role of candidate genes. Here we report a method for using transcription activator-like effector nucleases (TALENs) to mutate genes in surface A. mexicanus. Genome editing using TALENs in A. mexicanus has been utilized to generate mutations in pigmentation genes. This technique can also be utilized to evaluate the role of candidate genes for a number of other traits that have evolved in cave forms of A. mexicanus.

  19. Rh D blood group conversion using transcription activator-like effector nucleases

    PubMed Central

    Kim, Young-Hoon; Kim, Hyun O.; Baek, Eun J.; Kurita, Ryo; Cha, Hyuk-Jin; Nakamura, Yukio; Kim, Hyongbum

    2015-01-01

    Group O D-negative blood cells are universal donors in transfusion medicine and methods for converting other blood groups into this universal donor group have been researched. However, conversion of D-positive cells into D-negative is yet to be achieved, although conversion of group A or B cells into O cells has been reported. The Rh D blood group is determined by the RHD gene, which encodes a 12-transmembrane domain protein. Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs). After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained. Erythroid-lineage cells differentiated from these knockout erythroid progenitor cells do not agglutinate in the presence of anti-D reagents and do not express D antigen, as assessed using flow cytometry. Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine. PMID:26078220

  20. Generation of myostatin B knockout yellow catfish (Tachysurus fulvidraco) using transcription activator-like effector nucleases.

    PubMed

    Dong, Zhangji; Ge, Jiachun; Xu, Zhiqiang; Dong, Xiaohua; Cao, Shasha; Pan, Jianlin; Zhao, Qingshun

    2014-06-01

    Myostatin (Mstn), a member of the transforming growth factor β superfamily, plays an inhibiting role in mammalian muscle growth. Mammals like human, cattle, mouse, sheep, and dog carrying null alleles of Mstn display a double-muscle phenotype. Mstn is conserved in fish; however, little is known whether the fish with mutated mstn display a similar phenotype to mammals because of the lack of mutant fish with mstn null alleles. Previously, we knocked out one of the duplicated copies of myostatin gene (mstna) in yellow catfish using zinc-finger nucleases. In this study, we report the identification of the second myostatin gene (mstnb) and knockout of mstnb in yellow catfish. The gene comprises three exons. It is predicted to encode 373 amino acid residues. The predicted protein exhibits 59.3% identity with yellow catfish Mstna and 57.3% identity with human MSTN. Employing TALEN (transcription activator-like effector nucleases) technology, we obtained two founders (from four randomly selected founders) of yellow catfish carrying the mutated mstnb gene in their germ cells. Totally, six mutated alleles of mstnb were obtained from the founders. Among the six alleles, four are nonframeshift and two are frameshift mutation. The frameshift mutated alleles include mstnb(nju22), an 8 bp deletion, and mstnb(nju24), a complex type of mutation comprising a 7 bp deletion and a 12 bp insertion. They are predicted to encode function null Mstnb. Our results will help to understand the roles of mstn genes in fish growth.

  1. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases

    PubMed Central

    Qiu, Zhongwei; Liu, Meizhen; Chen, Zhaohua; Shao, Yanjiao; Pan, Hongjie; Wei, Gaigai; Yu, Chao; Zhang, Long; Li, Xia; Wang, Ping; Fan, Heng-Yu; Du, Bing; Liu, Bin; Liu, Mingyao; Li, Dali

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background. PMID:23630316

  2. Transcription Activator-Like Effector Nucleases (TALEN)-Mediated Targeted DNA Insertion in Potato Plants

    PubMed Central

    Forsyth, Adrienne; Weeks, Troy; Richael, Craig; Duan, Hui

    2016-01-01

    Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. Specifically integrated transgenes are guaranteed to co-segregate, and expression level is more predictable, which makes downstream characterization and line selection more manageable. Because the site of DNA integration is known, the steps to deregulation of transgenic crops may be simplified. Here we describe a method that combines transcription activator-like effector nuclease (TALEN)-mediated induction of double strand breaks (DSBs) and non-autonomous marker selection to insert a transgene into a pre-selected, transcriptionally active region in the potato genome. In our experiment, TALEN was designed to create a DSB in the genome sequence following an endogenous constitutive promoter. A cytokinin vector was utilized for TALENs expression and prevention of stable integration of the nucleases. The donor vector contained a gene of interest cassette and a promoter-less plant-derived herbicide resistant gene positioned near the T-DNA left border which was used to select desired transgenic events. Our results indicated that TALEN induced T-DNA integration occurred with high frequency and resulting events have consistent expression of the gene of interest. Interestingly, it was found that, in most lines integration took place through one sided homology directed repair despite the minimal homologous sequence at the right border. An efficient transient assay for TALEN activity verification is also described. PMID:27826306

  3. Transcription activator-like effector nucleases enable efficient plant genome engineering.

    PubMed

    Zhang, Yong; Zhang, Feng; Li, Xiaohong; Baller, Joshua A; Qi, Yiping; Starker, Colby G; Bogdanove, Adam J; Voytas, Daniel F

    2013-01-01

    The ability to precisely engineer plant genomes offers much potential for advancing basic and applied plant biology. Here, we describe methods for the targeted modification of plant genomes using transcription activator-like effector nucleases (TALENs). Methods were optimized using tobacco (Nicotiana tabacum) protoplasts and TALENs targeting the acetolactate synthase (ALS) gene. Optimal TALEN scaffolds were identified using a protoplast-based single-strand annealing assay in which TALEN cleavage creates a functional yellow fluorescent protein gene, enabling quantification of TALEN activity by flow cytometry. Single-strand annealing activity data for TALENs with different scaffolds correlated highly with their activity at endogenous targets, as measured by high-throughput DNA sequencing of polymerase chain reaction products encompassing the TALEN recognition sites. TALENs introduced targeted mutations in ALS in 30% of transformed cells, and the frequencies of targeted gene insertion approximated 14%. These efficiencies made it possible to recover genome modifications without selection or enrichment regimes: 32% of tobacco calli generated from protoplasts transformed with TALEN-encoding constructs had TALEN-induced mutations in ALS, and of 16 calli characterized in detail, all had mutations in one allele each of the duplicate ALS genes (SurA and SurB). In calli derived from cells treated with a TALEN and a 322-bp donor molecule differing by 6 bp from the ALS coding sequence, 4% showed evidence of targeted gene replacement. The optimized reagents implemented in plant protoplasts should be useful for targeted modification of cells from diverse plant species and using a variety of means for reagent delivery.

  4. Draft genome sequence of Xanthomonas axonopodis pv. glycines 8ra possessing transcription activator-like effectors used for genetic engineering.

    PubMed

    Lee, Ju-Hoon; Shin, Hakdong; Park, Hye-Jee; Ryu, Sangryeol; Han, Sang-Wook

    2014-06-10

    Xanthomonas axonopodis pv. glycines 8ra is a causal agent of bacterial pustule disease in soybean. This bacterium possesses transcription activator-like (TAL) effectors which are useful for genetic/protein engineering applications in higher organisms including plants and humans. Here, we report that the draft genome sequence consists of 5,337,885-bp double-stranded DNA encoding 4674 open reading frames (ORFs) in 13 different contigs. This genome sequence would be useful in applications of TAL effectors in genetic engineering and in elucidating virulence factors against plants.

  5. Generation of GFP Reporter Human Induced Pluripotent Stem Cells Using AAVS1 Safe Harbor Transcription Activator-Like Effector Nuclease.

    PubMed

    Luo, Yongquan; Rao, Mahendra; Zou, Jizhong

    2014-05-16

    Generation of a fluorescent GFP reporter line in human induced pluripotent stem cells (hiPSCs) provides enormous potentials in both basic stem cell research and regenerative medicine. A protocol for efficiently generating such an engineered reporter line by gene targeting is highly desired. Transcription activator-like effector nucleases (TALENs) are a new class of artificial restriction enzymes that have been shown to significantly promote homologous recombination by >1000-fold. The AAVS1 (adeno-associated virus integration site 1) locus is a "safe harbor" and has an open chromatin structure that allows insertion and stable expression of transgene. Here, we describe a step-by-step protocol from determination of TALENs activity, hiPSC culture, and delivery of a donor into AAVS1 targeting site, to validation of targeted integration by PCR and Southern blot analysis using hiPSC line, and a pair of open-source AAVS1 TALENs.

  6. A transcription activator-like effector from Xanthomonas oryzae pv. oryzicola elicits dose-dependent resistance in rice.

    PubMed

    Hummel, Aaron W; Wilkins, Katherine E; Wang, Li; Cernadas, R Andres; Bogdanove, Adam J

    2017-01-01

    Xanthomonas spp. reduce crop yields and quality worldwide. During infection of their plant hosts, many strains secrete transcription activator-like (TAL) effectors, which enter the host cell nucleus and activate specific corresponding host genes at effector binding elements (EBEs) in the promoter. TAL effectors may contribute to disease by activating the expression of susceptibility genes or trigger resistance associated with the hypersensitive reaction (HR) by activating an executor resistance (R) gene. The rice bacterial leaf streak pathogen X. oryzae pv. oryzicola (Xoc) is known to suppress host resistance, and no host R gene has been identified against it, despite considerable effort. To further investigate Xoc suppression of host resistance, we conducted a screen of effectors from BLS256 and identified Tal2a as an HR elicitor in rice when delivered heterologously by a strain of the closely related rice bacterial blight pathogen X. oryzae pv. oryzae (Xoo) or by the soybean pathogen X. axonopodis pv. glycines. The HR required the Tal2a activation domain, suggesting an executor R gene. Tal2a activity was differentially distributed among geographically diverse Xoc isolates, being largely conserved among Asian isolates. We identified four genes induced by Tal2a in next-generation RNA sequencing experiments and confirmed them using quantitative real-time reverse transcription-polymerase chain reaction (qPCR). However, neither individual nor collective activation of these genes by designer TAL effectors resulted in HR. A tal2a knockout mutant of BLS256 showed virulence comparable with the wild-type, but plasmid-based overexpression of tal2a at different levels in the wild-type reduced virulence in a directly corresponding way. Overall, the results reveal that host resistance suppression by Xoc plays a critical role in pathogenesis. Further, the dose-dependent avirulence activity of Tal2a and the apparent lack of a single canonical target that accounts for HR point to

  7. Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins

    PubMed Central

    Jia, Jingyue; Bai, Fang; Jin, Yongxin; Santostefano, Katherine E.; Ha, Un-Hwan; Wu, Donghai

    2015-01-01

    The type III secretion system (T3SS) of Pseudomonas aeruginosa is a powerful tool for direct protein delivery into mammalian cells and has successfully been used to deliver various exogenous proteins into mammalian cells. In the present study, transcription activator-like effector nuclease (TALEN) proteins have been efficiently delivered using the P. aeruginosa T3SS into mouse embryonic stem cells (mESCs), human ESCs (hESCs), and human induced pluripotent stem cells (hiPSCs) for genome editing. This bacterial delivery system offers an alternative method of TALEN delivery that is highly efficient in cleavage of the chromosomal target and presumably safer by avoiding plasmid DNA introduction. We combined the method of bacterial T3SS-mediated TALEN protein injection and transfection of an oligonucleotide template to effectively generate precise genetic modifications in the stem cells. Initially, we efficiently edited a single-base in the gfp gene of a mESC line to silence green fluorescent protein (GFP) production. The resulting GFP-negative mESC was cloned from a single cell and subsequently mutated back to a GFP-positive mESC line. Using the same approach, the gfp gene was also effectively knocked out in hESCs. In addition, a defined single-base edition was effectively introduced into the X-chromosome-linked HPRT1 gene in hiPSCs, generating an in vitro model of Lesch-Nyhan syndrome. T3SS-mediated TALEN protein delivery provides a highly efficient alternative for introducing precise gene editing within pluripotent stem cells for the purpose of disease genotype-phenotype relationship studies and cellular replacement therapies. Significance The present study describes a novel and powerful tool for the delivery of the genome editing enzyme transcription activator-like effector nuclease (TALEN) directly into pluripotent stem cells (PSCs), achieving desired base changes on the genomes of PSCs with high efficiency. This novel approach uses bacteria as a protein delivery

  8. Simultaneous gene editing by injection of mRNAs encoding transcription activator-like effector nucleases into mouse zygotes.

    PubMed

    Li, Chunliang; Qi, Rong; Singleterry, Rebecca; Hyle, Judith; Balch, Amanda; Li, Xiuling; Sublett, Jack; Berns, Hartmut; Valentine, Marcus; Valentine, Virginia; Sherr, Charles J

    2014-05-01

    Injection of transcription activator-like effector nucleases (TALEN) mRNAs into mouse zygotes transferred into foster mothers efficiently generated founder mice with heritable mutations in targeted genes. Immunofluorescence visualization of phosphorylated histone 2A (γH2AX) combined with fluorescence in situ hybridization revealed that TALEN pairs targeting the Agouti locus induced site-directed DNA breaks in zygotes within 6 h of injection, an activity that continued at reduced efficiency in two-cell embryos. TALEN-Agouti mRNAs injected into zygotes of brown FvB × C57BL/6 hybrid mice generated completely black pups, confirming that mutations were induced prior to, and/or early after, cell division. Founder mice, many of which were mosaic, transmitted altered Agouti alleles to F1 pups to yield an allelic series of mutant strains. Although mutations were targeted to "spacer" sequences flanked by TALEN binding sites, larger deletions that extended beyond the TALEN-binding sequences were also detected and were similarly inherited through the germ line. Zygotic coinjection of TALEN mRNAs directed to the Agouti, miR-205, and the Arf tumor suppressor loci yielded pups containing frequent and heritable mutations of two or three genes. Simultaneous gene editing in zygotes affords an efficient approach for producing mice with compound mutant phenotypes, bypassing constraints of conventional mouse knockout technology in embryonic stem cells.

  9. Design, evaluation, and screening methods for efficient targeted mutagenesis with transcription activator-like effector nucleases in medaka.

    PubMed

    Ansai, Satoshi; Inohaya, Keiji; Yoshiura, Yasutoshi; Schartl, Manfred; Uemura, Norihito; Takahashi, Ryosuke; Kinoshita, Masato

    2014-01-01

    Genome editing using engineered nucleases such as transcription activator-like effector nucleases (TALENs) has become a powerful technology for reverse genetics. In this study, we have described efficient detection methods for TALEN-induced mutations at endogenous loci and presented guidelines of TALEN design for efficient targeted mutagenesis in medaka, Oryzias latipes. We performed a heteroduplex mobility assay (HMA) using an automated microchip electrophoresis system, which is a simple and high-throughput method for evaluation of in vivo activity of TALENs and for genotyping mutant fish of F1 or later generations. We found that a specific pattern of mutations is dominant for TALENs harboring several base pairs of homologous sequences in target sequence. Furthermore, we found that a 5' T, upstream of each TALEN-binding sequence, is not essential for genomic DNA cleavage. Our findings provide information that expands the potential of TALENs and other engineered nucleases as tools for targeted genome editing in a wide range of organisms, including medaka.

  10. Generation of obese rat model by transcription activator-like effector nucleases targeting the leptin receptor gene.

    PubMed

    Chen, Yuting; Lu, Wenqing; Gao, Na; Long, Yi; Shao, Yanjiao; Liu, Meizhen; Chen, Huaqing; Ye, Shixin; Ma, Xueyun; Liu, Mingyao; Li, Dali

    2017-02-01

    The laboratory rat is a valuable mammalian model organism for basic research and drug discovery. Here we demonstrate an efficient methodology by applying transcription activator-like effector nucleases (TALENs) technology to generate Leptin receptor (Lepr) knockout rats on the Sprague Dawley (SD) genetic background. Through direct injection of in vitro transcribed mRNA of TALEN pairs into SD rat zygotes, somatic mutations were induced in two of three resulting pups. One of the founders carrying bi-allelic mutation exhibited early onset of obesity and infertility. The other founder carried a chimeric mutation which was efficiently transmitted to the progenies. Through phenotyping of the resulting three lines of rats bearing distinct mutations in the Lepr locus, we found that the strains with a frame-shifted or premature stop codon mutation led to obesity and metabolic disorders. However, no obvious defect was observed in a strain with an in-frame 57 bp deletion in the extracellular domain of Lepr. This suggests the deleted amino acids do not significantly affect Lepr structure and function. This is the first report of generating the Lepr mutant obese rat model in SD strain through a reverse genetic approach. This suggests that TALEN is an efficient and powerful gene editing technology for the generation of disease models.

  11. Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.

    PubMed

    Weyman, Philip D; Beeri, Karen; Lefebvre, Stephane C; Rivera, Josefa; McCarthy, James K; Heuberger, Adam L; Peers, Graham; Allen, Andrew E; Dupont, Christopher L

    2015-05-01

    Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.

  12. Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae.

    PubMed

    Aouida, Mustapha; Li, Lixin; Mahjoub, Ali; Alshareef, Sahar; Ali, Zahir; Piatek, Agnieszka; Mahfouz, Magdy M

    2015-10-01

    Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S. cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S. cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production.

  13. Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases.

    PubMed

    Sakane, Yuto; Sakuma, Tetsushi; Kashiwagi, Keiko; Kashiwagi, Akihiko; Yamamoto, Takashi; Suzuki, Ken-Ichi T

    2014-01-01

    Transcription activator-like effector nucleases (TALENs) have been extensively used in genome editing in various organisms. In some cases, however, it is difficult to efficiently disrupt both paralogous genes using a single pair of TALENs in Xenopus laevis because of its polyploidy. Here, we report targeted mutagenesis of multiple and paralogous genes using two pairs of TALENs in X. laevis. First, we show simultaneous targeted mutagenesis of three genes, tyrosinase paralogues (tyra and tyrb) and enhanced green fluorescent protein (egfp) by injection of two TALENs pairs in transgenic embryos carrying egfp. Consistent with the high frequency of both severe phenotypic traits, albinism and loss of GFP fluorescence, frameshift mutation rates of tyr paralogues and egfp reached 40-80%. Next, we show early introduction of TALEN-mediated mutagenesis of these target loci during embryogenesis. Finally, we also demonstrate that two different pairs of TALENs can simultaneously introduce mutations to both paralogues encoding histone chaperone with high efficiency. Our results suggest that targeted mutagenesis of multiple genes using TALENs can be applied to analyze the functions of paralogous genes with redundancy in X. laevis.

  14. Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases.

    PubMed

    Sommer, Daniel; Peters, Annika; Wirtz, Tristan; Mai, Maren; Ackermann, Justus; Thabet, Yasser; Schmidt, Jürgen; Weighardt, Heike; Wunderlich, F Thomas; Degen, Joachim; Schultze, Joachim L; Beyer, Marc

    2014-01-01

    Generation of mouse models by introducing transgenes using homologous recombination is critical for understanding fundamental biology and pathology of human diseases. Here we investigate whether artificial transcription activator-like effector nucleases (TALENs)-powerful tools that induce DNA double-strand breaks at specific genomic locations-can be combined with a targeting vector to induce homologous recombination for the introduction of a transgene in embryonic stem cells and fertilized murine oocytes. We describe the generation of a conditional mouse model using TALENs, which introduce double-strand breaks at the genomic locus of the special AT-rich sequence-binding protein-1 in combination with a large 14.4 kb targeting template vector. We report successful germline transmission of this allele and demonstrate its recombination in primary cells in the presence of Cre-recombinase. These results suggest that TALEN-assisted induction of DNA double-strand breaks can facilitate homologous recombination of complex targeting constructs directly in oocytes.

  15. An efficient antiviral strategy for targeting hepatitis B virus genome using transcription activator-like effector nucleases.

    PubMed

    Chen, Jieliang; Zhang, Wen; Lin, Junyu; Wang, Fan; Wu, Min; Chen, Cuncun; Zheng, Ye; Peng, Xiuhua; Li, Jianhua; Yuan, Zhenghong

    2014-02-01

    The hepatitis B virus (HBV) is a DNA virus that can cause chronic hepatitis B (CHB) in humans. Current therapies for CHB infection are limited in efficacy and do not target the pre-existing viral genomic DNA, which are present in the nucleus as a covalently closed circular DNA (cccDNA) form. The transcription activator-like (TAL) effector nucleases (TALENs) are newly developed enzymes that can cleave sequence-specific DNA targets. Here, TALENs targeting the conserved regions of the viral genomic DNA among different HBV genotypes were constructed. The expression of TALENs in Huh7 cells transfected with monomeric linear full-length HBV DNA significantly reduced the viral production of HBeAg, HBsAg, HBcAg, and pgRNA, resulted in a decreased cccDNA level and misrepaired cccDNAs without apparent cytotoxic effects. The anti-HBV effect of TALENs was further demonstrated in a hydrodynamic injection-based mouse model. In addition, an enhanced antiviral effect with combinations of TALENs and interferon-α (IFN-α) treatment was observed and expression of TALENs restored HBV suppressed IFN-stimulated response element-directed transcription. Taken together, these data indicate that TALENs can specifically target and successfully inactivate the HBV genome and are potently synergistic with IFN-α, thus providing a potential therapeutic strategy for treating CHB infection.

  16. Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases.

    PubMed

    Bloom, Kristie; Ely, Abdullah; Mussolino, Claudio; Cathomen, Toni; Arbuthnot, Patrick

    2013-10-01

    Chronic hepatitis B virus (HBV) infection remains an important global health problem. Stability of the episomal covalently closed circular HBV DNA (cccDNA) is largely responsible for the modest curative efficacy of available therapy. Since licensed anti-HBV drugs have a post-transcriptional mechanism of action, disabling cccDNA is potentially of therapeutic benefit. To develop this approach, we engineered mutagenic transcription activator-like effector nucleases (TALENs) that target four HBV-specific sites within the viral genome. TALENs with cognate sequences in the S or C open-reading frames (ORFs) efficiently disrupted sequences at the intended sites and suppressed markers of viral replication. Following triple transfection of cultured HepG2.2.15 cells under mildly hypothermic conditions, the S TALEN caused targeted mutation in ~35% of cccDNA molecules. Markers of viral replication were also inhibited in vivo in a murine hydrodynamic injection model of HBV replication. HBV target sites within S and C ORFs of the injected HBV DNA were mutated without evidence of toxicity. These findings are the first to demonstrate a targeted nuclease-mediated disruption of HBV cccDNA. Efficacy in vivo also indicates that these engineered nucleases have potential for use in treatment of chronic HBV infection.

  17. DNA Methylation Affects the Efficiency of Transcription Activator-Like Effector Nucleases-Mediated Genome Editing in Rice

    PubMed Central

    Kaya, Hidetaka; Numa, Hisataka; Nishizawa-Yokoi, Ayako; Toki, Seiichi; Habu, Yoshiki

    2017-01-01

    Genome editing in plants becomes popular since the advent of sequence-specific nucleases (SSNs) that are simple to set up and efficient in various plant species. Although transcription activator-like effector nucleases (TALENs) are one of the most prevalent SSNs and have a potential to provide higher target specificity by their dimeric property, TALENs are sensitive to methylated cytosines that are present not only in transposons but also in active genes in plants. In mammalian cells, the methylation sensitivity of TALENs could be overcome by using a base-recognition module (N∗) that has a higher affinity to methylated cytosine. In contrast to mammals, plants carry DNA methylation at all cytosine contexts (CG, CHG, and CHH, where H represents A, C, or T) with various degrees and effectiveness of N∗ module in genome editing in plants has not been explored. In this study, we designed sets of TALENs with or without N∗ modules and examined their efficiency in genome editing of methylated regions in rice. Although improvement in genome editing efficiency was observed with N∗-TALENs designed to a stably methylated target, another target carrying cytosines with various levels of methylation showed resistance to both normal and N∗-TALENs. The results suggest that variability of cytosine methylation in target regions is an additional factor affecting the genome editing efficiency of TALENs. PMID:28348570

  18. TALE-Like Effectors Are an Ancestral Feature of the Ralstonia solanacearum Species Complex and Converge in DNA Targeting Specificity

    PubMed Central

    Schandry, Niklas; de Lange, Orlando; Prior, Philippe; Lahaye, Thomas

    2016-01-01

    Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that transcription activator-like (TAL) effectors from Ralstonia, termed RipTALs, are capable of activating reporter genes in planta, if these are preceded by a matching effector binding element (EBE). RipTALs target DNA via their central repeat domain (CRD), where one repeat pairs with one DNA-base of the given EBE. The repeat variable diresidue dictates base repeat specificity in a predictable fashion, known as the TALE code. In this work, we analyze RipTALs across all phylotypes of the Ralstonia solanacearum species complex. We find that RipTALs are prevalent in phylotypes I and IV but absent from most phylotype III and II strains (10/12, 8/14, 1/24, and 1/5 strains contained a RipTAL, respectively). RipTALs originating from strains of the same phylotype show high levels of sequence similarity (>98%) in the N-terminal and C-terminal regions, while RipTALs isolated from different phylotypes show 47–91% sequence similarity in those regions, giving rise to four RipTAL classes. We show that, despite sequence divergence, the base preference for guanine, mediated by the N-terminal region, is conserved across RipTALs of all classes. Using the number and order of repeats found in the CRD, we functionally sub-classify RipTALs, introduce a new simple nomenclature, and predict matching EBEs for all seven distinct RipTALs identified. We experimentally study RipTAL EBEs and uncover that some RipTALs are able to target the EBEs of other RipTALs, referred to as cross-reactivity. In particular, RipTALs from strains with a broad host range on solanaceous hosts cross-react on each other’s EBEs. Investigation of sequence divergence

  19. AnnoTALE: bioinformatics tools for identification, annotation, and nomenclature of TALEs from Xanthomonas genomic sequences

    PubMed Central

    Grau, Jan; Reschke, Maik; Erkes, Annett; Streubel, Jana; Morgan, Richard D.; Wilson, Geoffrey G.; Koebnik, Ralf; Boch, Jens

    2016-01-01

    Transcription activator-like effectors (TALEs) are virulence factors, produced by the bacterial plant-pathogen Xanthomonas, that function as gene activators inside plant cells. Although the contribution of individual TALEs to infectivity has been shown, the specific roles of most TALEs, and the overall TALE diversity in Xanthomonas spp. is not known. TALEs possess a highly repetitive DNA-binding domain, which is notoriously difficult to sequence. Here, we describe an improved method for characterizing TALE genes by the use of PacBio sequencing. We present ‘AnnoTALE’, a suite of applications for the analysis and annotation of TALE genes from Xanthomonas genomes, and for grouping similar TALEs into classes. Based on these classes, we propose a unified nomenclature for Xanthomonas TALEs that reveals similarities pointing to related functionalities. This new classification enables us to compare related TALEs and to identify base substitutions responsible for the evolution of TALE specificities. PMID:26876161

  20. Single molecule real-time sequencing of Xanthomonas oryzae genomes reveals a dynamic structure and complex TAL (transcription activator-like) effector gene relationships

    PubMed Central

    Booher, Nicholas J.; Carpenter, Sara C. D.; Sebra, Robert P.; Wang, Li; Salzberg, Steven L.; Leach, Jan E.; Bogdanove, Adam J.

    2016-01-01

    Pathogen-injected, direct transcriptional activators of host genes, TAL (transcription activator-like) effectors play determinative roles in plant diseases caused by Xanthomonas spp. A large domain of nearly identical, 33–35 aa repeats in each protein mediates DNA recognition. This modularity makes TAL effectors customizable and thus important also in biotechnology. However, the repeats render TAL effector (tal) genes nearly impossible to assemble using next-generation, short reads. Here, we demonstrate that long-read, single molecule real-time (SMRT) sequencing solves this problem. Taking an ensemble approach to first generate local, tal gene contigs, we correctly assembled de novo the genomes of two strains of the rice pathogen X. oryzae completed previously using the Sanger method and even identified errors in those references. Sequencing two more strains revealed a dynamic genome structure and a striking plasticity in tal gene content. Our results pave the way for population-level studies to inform resistance breeding, improve biotechnology and probe TAL effector evolution. PMID:27148456

  1. Gene targeting technologies in rats: zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats.

    PubMed

    Mashimo, Tomoji

    2014-01-01

    The laboratory rat has been widely used as an animal model in biomedical science for more than 150 years. Applying zinc-finger nucleases or transcription activator-like effector nucleases to rat embryos via microinjection is an efficient genome editing tool for generating targeted knockout rats. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonucleases have been used as an effective tool for precise and multiplex genome editing in mice and rats. In this review, the advantages and disadvantages of these site-specific nuclease technologies for genetic analysis and manipulation in rats are discussed.

  2. Targeting DNA double-strand breaks with TAL effector nucleases.

    PubMed

    Christian, Michelle; Cermak, Tomas; Doyle, Erin L; Schmidt, Clarice; Zhang, Feng; Hummel, Aaron; Bogdanove, Adam J; Voytas, Daniel F

    2010-10-01

    Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.

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

    PubMed

    Gersbach, Charles A; Perez-Pinera, Pablo

    2014-08-01

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

  4. Transcription activator-like effector nucleases efficiently disrupt the target gene in Iberian ribbed newts (Pleurodeles waltl), an experimental model animal for regeneration.

    PubMed

    Hayashi, Toshinori; Sakamoto, Kousuke; Sakuma, Tetsushi; Yokotani, Naoki; Inoue, Takeshi; Kawaguchi, Eri; Agata, Kiyokazu; Yamamoto, Takashi; Takeuchi, Takashi

    2014-01-01

    Regeneration of a lost tissue in an animal is an important issue. Although regenerative studies have a history of research spanning more than a century, the gene functions underlying regulation of the regeneration are mostly unclear. Analysis of knockout animals is a very powerful tool with which to elucidate gene function. Recently, transcription activator-like effector nucleases (TALENs) have been developed as an effective technique for genome editing. This technique enables gene targeting in amphibians such as newts that were previously impossible. Here we show that newts microinjected with TALEN mRNAs designed for targeting the tyrosinase gene in single-cell stage embryos revealed an albino phenotype. Sequence analysis revealed that the tyrosinase genes were effectively disrupted in these albino newts. Moreover, precise genome alteration was achieved using TALENs and single strand oligodeoxyribonucleotides. Our results suggest that TALENs are powerful tools for genome editing for regenerative research in newts.

  5. TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-Mediated Generation and Metabolic Analysis of Camalexin-Deficient cyp71a12 cyp71a13 Double Knockout Lines.

    PubMed

    Müller, Teresa M; Böttcher, Christoph; Morbitzer, Robert; Götz, Cornelia C; Lehmann, Johannes; Lahaye, Thomas; Glawischnig, Erich

    2015-07-01

    In Arabidopsis (Arabidopsis thaliana), a number of defense-related metabolites are synthesized via indole-3-acetonitrile (IAN), including camalexin and indole-3-carboxylic acid (ICOOH) derivatives. Cytochrome P450 71A13 (CYP71A13) is a key enzyme for camalexin biosynthesis and catalyzes the conversion of indole-3-acetaldoxime (IAOx) to IAN. The CYP71A13 gene is located in tandem with its close homolog CYP71A12, also encoding an IAOx dehydratase. However, for CYP71A12, indole-3-carbaldehyde and cyanide were identified as major reaction products. To clarify CYP71A12 function in vivo and to better understand IAN metabolism, we generated two cyp71a12 cyp71a13 double knockout mutant lines. CYP71A12-specific transcription activator-like effector nucleases were introduced into the cyp71a13 background, and very efficient somatic mutagenesis was achieved. We observed stable transmission of the cyp71a12 mutation to the following generations, which is a major challenge for targeted mutagenesis in Arabidopsis. In contrast to cyp71a13 plants, in which camalexin accumulation is partially reduced, double mutants synthesized only traces of camalexin, demonstrating that CYP71A12 contributes to camalexin biosynthesis in leaf tissue. A major role of CYP71A12 was identified for the inducible biosynthesis of ICOOH. Specifically, the ICOOH methyl ester was reduced to 12% of the wild-type level in AgNO3-challenged cyp71a12 leaves. In contrast, indole-3-carbaldehyde derivatives apparently are synthesized via alternative pathways, such as the degradation of indole glucosinolates. Based on these results, we present a model for this surprisingly complex metabolic network with multiple IAN sources and channeling of IAOx-derived IAN into camalexin biosynthesis. In conclusion, transcription activator-like effector nuclease-mediated mutation is a powerful tool for functional analysis of tandem genes in secondary metabolism.

  6. Interfering TAL effectors of Xanthomonas oryzae neutralize R-gene-mediated plant disease resistance

    PubMed Central

    Ji, Zhiyuan; Ji, Chonghui; Liu, Bo; Zou, Lifang; Chen, Gongyou; Yang, Bing

    2016-01-01

    Plant pathogenic bacteria of the genus Xanthomonas possess transcription activator-like effectors (TALEs) that activate transcription of disease susceptibility genes in the host, inducing a state of disease. Here we report that some isolates of the rice pathogen Xanthomonas oryzae use truncated versions of TALEs (which we term interfering TALEs, or iTALEs) to overcome disease resistance. In comparison with typical TALEs, iTALEs lack a transcription activation domain but retain nuclear localization motifs and are expressed from genes that were previously considered pseudogenes. We show that the rice gene Xa1, encoding a nucleotide-binding leucine-rich repeat protein, confers resistance against X. oryzae isolates by recognizing multiple TALEs. However, the iTALEs present in many isolates interfere with the otherwise broad-spectrum resistance conferred by Xa1. Our findings illustrate how bacterial effectors that trigger disease resistance in the host can evolve to interfere with the resistance process and, thus, promote disease. PMID:27811915

  7. Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology.

    PubMed

    Kang, Jung-Taek; Kwon, Dae-Kee; Park, A-Rum; Lee, Eun-Jin; Yun, Yun-Jin; Ji, Dal-Young; Lee, Kiho; Park, Kwang-Wook

    2016-03-01

    Recent developments in genome editing technology using meganucleases demonstrate an efficient method of producing gene edited pigs. In this study, we examined the effectiveness of the transcription activator-like effector nuclease (TALEN) system in generating specific mutations on the pig genome. Specific TALEN was designed to induce a double-strand break on exon 9 of the porcine α1,3-galactosyltransferase (GGTA1) gene as it is the main cause of hyperacute rejection after xenotransplantation. Human decay-accelerating factor (hDAF) gene, which can produce a complement inhibitor to protect cells from complement attack after xenotransplantation, was also integrated into the genome simultaneously. Plasmids coding for the TALEN pair and hDAF gene were transfected into porcine cells by electroporation to disrupt the porcine GGTA1 gene and express hDAF. The transfected cells were then sorted using a biotin-labeled IB4 lectin attached to magnetic beads to obtain GGTA1 deficient cells. As a result, we established GGTA1 knockout (KO) cell lines with biallelic modification (35.0%) and GGTA1 KO cell lines expressing hDAF (13.0%). When these cells were used for somatic cell nuclear transfer, we successfully obtained live GGTA1 KO pigs expressing hDAF. Our results demonstrate that TALEN-mediated genome editing is efficient and can be successfully used to generate gene edited pigs.

  8. Knockout of a transgene by transcription activator-like effector nucleases (TALENs) in the sawfly, Athalia rosae (Hymenoptera) and the ladybird beetle, Harmonia axyridis (Coleoptera).

    PubMed

    Hatakeyama, M; Yatomi, J; Sumitani, M; Takasu, Y; Sekiné, K; Niimi, T; Sezutsu, H

    2016-02-01

    Transcription activator-like effector nucleases (TALENs) are efficient tools for targeted genome editing and have been utilized in a number of insects. Here, we demonstrate the gene disruption (knockout) caused by TALENs targeting a transgene, 3xP3-driven enhanced green fluorescence protein (EGFP), that is integrated in the genome of two species, the sawfly Athalia rosae (Hymenoptera) and the ladybird beetle Harmonia axyridis (Coleoptera). Messenger RNAs of TALENs targeting the sequences adjacent to the chromophore region were microinjected into the eggs/embryos of each species. In At. rosae, when microinjection was performed at the posterior end of eggs, 15% of G(0) individuals showed a somatic mosaic phenotype for eye EGFP fluorescence. Three-quarters of the somatic mosaics produced EGFP-negative G(1) progeny. When eggs were injected at the anterior end, 63% of the G(0) individuals showed somatic mosaicism, and 17% of them produced EGFP-negative G(1) progeny. In H. axyridis, 25% of posterior-injected and 8% of anterior-injected G(0) individuals produced EGFP-negative G(1) progeny. In both species, the EGFP-negative progeny retained the EGFP gene, and various deletions were detected in the target sequences, indicating that gene disruption was successfully induced. Finally, for both species, 18-21% of G(0) founders produced gene knockout progeny sufficient for establishing knockout strains.

  9. The broad bacterial blight resistance of rice line CBB23 is triggered by a novel transcription activator-like (TAL) effector of Xanthomonas oryzae pv. oryzae.

    PubMed

    Wang, Chun-Lian; Qin, Teng-Fei; Yu, Hong-Man; Zhang, Xiao-Ping; Che, Jin-Ying; Gao, Ying; Zheng, Chong-Ke; Yang, Bing; Zhao, Kai-Jun

    2014-05-01

    Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is not only a disease devastating rice production worldwide, but also an ideal model system for the study of the interaction between plants and their bacterial pathogens. The rice near-isogenic line (NIL) CBB23, derived from a cross between a wild rice Oryza rufipogon accession (RBB16) and a susceptible indica rice variety (Jingang 30), is highly resistant to all field Xoo strains tested so far. Although the BB resistance of CBB23 has been widely used in rice breeding programmes, the mechanism of its extremely broad-spectrum resistance remains unknown. Here, we report the molecular cloning of an avirulence gene, designated as avrXa23, from Xoo strain PXO99(A) . We validate that AvrXa23, a novel transcription activator-like effector, specifically triggers the broad-spectrum BB resistance in CBB23. The prevalence of avrXa23 in all 38 Xoo strains surveyed may explain the broad-spectrum feature of BB resistance in CBB23. The results will significantly facilitate the molecular cloning of the corresponding resistance (R) gene in the host, and provide new insights into our understanding of the molecular mechanism for broad-spectrum disease resistance in plants.

  10. Knockout of the adp gene related with colonization in Bacillus nematocida B16 using customized transcription activator-like effectors nucleases

    PubMed Central

    Niu, Qiuhong; Zheng, Haoying; Zhang, Lin; Qin, Fujun; Facemire, Loryn; Zhang, Guo; Cao, Feng; Zhang, Ke-qin; Huang, Xiaowei; Yang, Jianwei; He, Lei; Liu, Chanjuan

    2015-01-01

    Bacillus nematocida B16 is able to dominate in the intestines of the worm Caenorhabditis elegans in ‘Trojan horse’ pathogenic mechanism. The adp is one candidate gene which potentially play a vital role in the colonization from our previous random mutagenesis screening results. To analyse the functional role of this gene, we constructed the adp knockout mutant through customized transcription activator-like effectors nucleases (TALEN), which has been successfully used in yeasts, nematodes, zebrafish and human pluripotent cells. Here, we first time report this knockout method in bacteria on this paper. Bioassay experiments demonstrated that the adp knockout mutant of B16 showed considerably lower colonization activity, reduced numbers of intestines and less than 80% nematocidal activity compared with the wild-type strain when infected for 48 h. However, no obvious change on proteolytic activity was observed in the mutant. Conversely, the complementation of adp gene restored most of the above deficient phenotypes. These results indicated that the adp gene was involved in surface adhesion and played a comparatively important role in colonizing host nematodes. Moreover, TALENs successfully disrupt target genes in bacteria. PMID:25912819

  11. Stable enhanced green fluorescent protein expression after differentiation and transplantation of reporter human induced pluripotent stem cells generated by AAVS1 transcription activator-like effector nucleases.

    PubMed

    Luo, Yongquan; Liu, Chengyu; Cerbini, Trevor; San, Hong; Lin, Yongshun; Chen, Guokai; Rao, Mahendra S; Zou, Jizhong

    2014-07-01

    Human induced pluripotent stem (hiPS) cell lines with tissue-specific or ubiquitous reporter genes are extremely useful for optimizing in vitro differentiation conditions as well as for monitoring transplanted cells in vivo. The adeno-associated virus integration site 1 (AAVS1) locus has been used as a "safe harbor" locus for inserting transgenes because of its open chromatin structure, which permits transgene expression without insertional mutagenesis. However, it is not clear whether targeted transgene expression at the AAVS1 locus is always protected from silencing when driven by various promoters, especially after differentiation and transplantation from hiPS cells. In this paper, we describe a pair of transcription activator-like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Using these TALENs for targeted gene addition, we find that the cytomegalovirus-immediate early enhancer/chicken β-actin/rabbit β-globin (CAG) promoter is better than cytomegalovirus 7 and elongation factor 1α short promoters in driving strong expression of the transgene. The two independent AAVS1, CAG, and enhanced green fluorescent protein (EGFP) hiPS cell reporter lines that we have developed do not show silencing of EGFP either in undifferentiated hiPS cells or in randomly and lineage-specifically differentiated cells or in teratomas. Transplanting cardiomyocytes from an engineered AAVS1-CAG-EGFP hiPS cell line in a myocardial infarcted mouse model showed persistent expression of the transgene for at least 7 weeks in vivo. Our results show that high-efficiency targeting can be obtained with open-source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo.

  12. Generation of Foxo3-targeted Mice by Injection of mRNAs Encoding Transcription Activator-like Effector Nucleases (TALENs) into Zygotes.

    PubMed

    Zhu, P; Liu, Q; Liu, S; Su, X; Feng, W; Lei, X; Liu, J; Cui, K; Huang, B; Shi, D

    2015-06-01

    In this study, for exploring the mechanism of forkhead box O3(Foxo3) participating in regulation of the activation of primordial oocytes, Foxo3-targeted mice were generated by injection of mRNAs encoding transcription activator-like effector nucleases (TALENs) into mouse zygotes. The TALEN sites were designed with high conservative homologous region among pig, bovine, buffalo and mouse by commercial bio-companies. The TALENs mutagenic non-homologous end-joining (NHEJ) repair activity were determined to be 31.3% in human embryonic kidney 293T (HEK-293T) cells by dual luciferase reporter assay system. Then, we firstly injected TALEN-mRNAs into the cytoplasm of mouse zygotes by micromanipulation, and four of 48 mouse blastocysts were identified as mutation by sequencing. Subsequently, by the method of TALEN-mRNAs injected into the zygotes with pronucleus micromanipulation technique, we obtained seven Foxo3 mutants of 20 FVB/NJ backgrounds mice which were Foxo3-independent alleles with frameshift and deletion mutations. It was very interesting that all seven were heterozygous mutants (Foxo3(-/+) ), and the gene mutagenesis rates of the mice reached 35%. The five Foxo3 mutant females were all infertile in the following 6 months after birth. The histological examination results showed that there were rare primordial follicles and primary follicles in the ovary of Foxo3 mutant compared to that of wide-type female mice. Moreover, one of two mutant males was subfertile and another was fertile normally. Those results suggested that the mutant of Foxo3 severely affected the fertile ability of female and perhaps male in some degree; furthermore, an even more efficient TALENs-based gene mutation method has been established to be poised to revolutionize the study of mouse and other species genetics.

  13. Targeting of the Plzf Gene in the Rat by Transcription Activator-Like Effector Nuclease Results in Caudal Regression Syndrome in Spontaneously Hypertensive Rats.

    PubMed

    Liška, František; Peterková, Renata; Peterka, Miroslav; Landa, Vladimír; Zídek, Václav; Mlejnek, Petr; Šilhavý, Jan; Šimáková, Miroslava; Křen, Vladimír; Starker, Colby G; Voytas, Daniel F; Izsvák, Zsuzsanna; Pravenec, Michal

    2016-01-01

    Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases). SHR ova were microinjected with constructs pTAL438/439 coding for a sequence-specific endonuclease that binds to target sequence in the first coding exon of the Plzf gene. Out of 43 animals born after microinjection, we detected a single male founder. Sequence analysis revealed a deletion of G that resulted in frame shift mutation starting in codon 31 and causing a premature stop codon at position of amino acid 58. The Plzftm1Ipcv allele is semi-lethal since approximately 95% of newborn homozygous animals died perinatally. All homozygous animals exhibited manifestations of a caudal regression syndrome including tail anomalies and serious size reduction and deformities of long bones, and oligo- or polydactyly on the hindlimbs. The heterozygous animals only exhibited the tail anomalies. Impaired development of the urinary tract was also revealed: one homozygous and one heterozygous rat exhibited a vesico-ureteric reflux with enormous dilatation of ureters and renal pelvis. In the homozygote, this was combined with a hypoplastic kidney. These results provide evidence for the important role of Plzf gene during development of the caudal part of a body-column vertebrae, hindlimbs and urinary system in the rat.

  14. Targeting of the Plzf Gene in the Rat by Transcription Activator-Like Effector Nuclease Results in Caudal Regression Syndrome in Spontaneously Hypertensive Rats

    PubMed Central

    Liška, František; Peterková, Renata; Peterka, Miroslav; Landa, Vladimír; Zídek, Václav; Mlejnek, Petr; Šilhavý, Jan; Šimáková, Miroslava; Křen, Vladimír; Starker, Colby G.; Voytas, Daniel F.; Izsvák, Zsuzsanna; Pravenec, Michal

    2016-01-01

    Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases). SHR ova were microinjected with constructs pTAL438/439 coding for a sequence-specific endonuclease that binds to target sequence in the first coding exon of the Plzf gene. Out of 43 animals born after microinjection, we detected a single male founder. Sequence analysis revealed a deletion of G that resulted in frame shift mutation starting in codon 31 and causing a premature stop codon at position of amino acid 58. The Plzftm1Ipcv allele is semi-lethal since approximately 95% of newborn homozygous animals died perinatally. All homozygous animals exhibited manifestations of a caudal regression syndrome including tail anomalies and serious size reduction and deformities of long bones, and oligo- or polydactyly on the hindlimbs. The heterozygous animals only exhibited the tail anomalies. Impaired development of the urinary tract was also revealed: one homozygous and one heterozygous rat exhibited a vesico-ureteric reflux with enormous dilatation of ureters and renal pelvis. In the homozygote, this was combined with a hypoplastic kidney. These results provide evidence for the important role of Plzf gene during development of the caudal part of a body—column vertebrae, hindlimbs and urinary system in the rat. PMID:27727328

  15. Addition of transcription activator-like effector binding sites to a pathogen strain-specific rice bacterial blight resistance gene makes it effective against additional strains and against bacterial leaf streak.

    PubMed

    Hummel, Aaron W; Doyle, Erin L; Bogdanove, Adam J

    2012-09-01

    Xanthomonas transcription activator-like (TAL) effectors promote disease in plants by binding to and activating host susceptibility genes. Plants counter with TAL effector-activated executor resistance genes, which cause host cell death and block disease progression. We asked whether the functional specificity of an executor gene could be broadened by adding different TAL effector binding elements (EBEs) to it. We added six EBEs to the rice Xa27 gene, which confers resistance to strains of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) that deliver the TAL effector AvrXa27. The EBEs correspond to three other effectors from Xoo strain PXO99(A) and three from strain BLS256 of the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Stable integration into rice produced healthy lines exhibiting gene activation by each TAL effector, and resistance to PXO99(A) , a PXO99(A) derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains virulent toward wildtype Xa27 plants. Transcripts initiated primarily at a common site. Sequences in the EBEs were found to occur nonrandomly in rice promoters, suggesting an overlap with endogenous regulatory sequences. Thus, executor gene specificity can be broadened by adding EBEs, but caution is warranted because of the possible coincident introduction of endogenous regulatory elements.

  16. STAR: a simple TAL effector assembly reaction using isothermal assembly

    PubMed Central

    Gogolok, Sabine; Garcia-Diaz, Claudia; Pollard, Steven M.

    2016-01-01

    Transcription activator-like effectors (TALEs) contain modular programmable DNA binding domains. Fusing TALEs with effector domains creates synthetic transcription factors (TALE-TFs) or nucleases (TALENs), enabling precise gene manipulations. The construction of TALEs remains challenging due to their repetitive sequences. Here we report a simple TALE assembly reaction (STAR) that enables individual laboratories to generate multiple TALEs in a facile manner. STAR uses an isothermal assembly (‘Gibson assembly’) that is labour- and cost-effective, accessible, rapid and scalable. A small 68-part fragment library is employed, and the specific TALE repeat sequence is generated within ~8 hours. Sequence-verified TALENs or TALE-TF plasmids targeting 17 bp target sequences can be produced within three days, without the need for stepwise intermediate plasmid production. We demonstrate the utility of STAR through production of functional TALE-TFs capable of activating human SOX2 expression. STAR addresses some of the shortcomings of existing Golden Gate or solid-phase assembly protocols and enables routine production of TALE-TFs that will complement emerging CRISPR/Cas9-based reagents across diverse applications in mammalian stem cell and synthetic biology. PMID:27615025

  17. Direct observation of TALE protein dynamics reveals a two-state search mechanism

    PubMed Central

    Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M.

    2015-01-01

    Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins for which the fundamental mechanisms governing the search process are not fully understood. Here we use single-molecule techniques to directly observe TALE search dynamics along DNA templates. We find that TALE proteins are capable of rapid diffusion along DNA using a combination of sliding and hopping behaviour, which suggests that the TALE search process is governed in part by facilitated diffusion. We also observe that TALE proteins exhibit two distinct modes of action during the search process—a search state and a recognition state—facilitated by different subdomains in monomeric TALE proteins. Using TALE truncation mutants, we further demonstrate that the N-terminal region of TALEs is required for the initial non-specific binding and subsequent rapid search along DNA, whereas the central repeat domain is required for transitioning into the site-specific recognition state. PMID:26027871

  18. Direct observation of TALE protein dynamics reveals a two-state search mechanism

    NASA Astrophysics Data System (ADS)

    Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M.

    2015-06-01

    Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins for which the fundamental mechanisms governing the search process are not fully understood. Here we use single-molecule techniques to directly observe TALE search dynamics along DNA templates. We find that TALE proteins are capable of rapid diffusion along DNA using a combination of sliding and hopping behaviour, which suggests that the TALE search process is governed in part by facilitated diffusion. We also observe that TALE proteins exhibit two distinct modes of action during the search process--a search state and a recognition state--facilitated by different subdomains in monomeric TALE proteins. Using TALE truncation mutants, we further demonstrate that the N-terminal region of TALEs is required for the initial non-specific binding and subsequent rapid search along DNA, whereas the central repeat domain is required for transitioning into the site-specific recognition state.

  19. Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors

    PubMed Central

    Thakore, Pratiksha I.; Gersbach, Charles A.

    2016-01-01

    Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy. PMID:26443215

  20. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

    PubMed Central

    Nerys-Junior, Arildo; Costa, Lendel C.; Braga-Dias, Luciene P.; Oliveira, Márcia; Rossi, Átila D.; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S.; Tanuri, Amilcar

    2014-01-01

    Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity. PMID:24688299

  1. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases.

    PubMed

    Nerys-Junior, Arildo; Costa, Lendel C; Braga-Dias, Luciene P; Oliveira, Márcia; Rossi, Atila D; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S; Tanuri, Amilcar

    2014-03-01

    Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.

  2. Designer TAL effectors induce disease susceptibility and resistance to Xanthomonas oryzae pv. oryzae in rice.

    PubMed

    Li, Ting; Huang, Sheng; Zhou, Junhui; Yang, Bing

    2013-05-01

    TAL (transcription activator-like) effectors from Xanthomonas bacteria activate the cognate host genes, leading to disease susceptibility or resistance dependent on the genetic context of host target genes. The modular nature and DNA recognition code of TAL effectors enable custom-engineering of designer TAL effectors (dTALE) for gene activation. However, the feasibility of dTALEs as transcription activators for gene functional analysis has not been demonstrated. Here, we report the use of dTALEs, as expressed and delivered by the pathogenic Xanthomonas oryzae pv. oryzae (Xoo), in revealing the new function of two previously identified disease-related genes and the potential of one developmental gene for disease susceptibility in rice/Xoo interactions. The dTALE gene dTALE-xa27, designed to target the susceptible allele of the resistance gene Xa27, elicited a resistant reaction in the otherwise susceptible rice cultivar IR24. Four dTALE genes were made to induce the four annotated Xa27 homologous genes in rice cultivar Nipponbare, but none of the four induced Xa27-like genes conferred resistance to the dTALE-containing Xoo strains. A dTALE gene was also generated to activate the recessive resistance gene xa13, an allele of the disease-susceptibility gene Os8N3 (also named Xa13 or OsSWEET11, a member of sucrose efflux transporter SWEET gene family). The induction of xa13 by the dTALE rendered the resistant rice IRBB13 (xa13/xa13) susceptible to Xoo. Finally, OsSWEET12, an as-yet uncharacterized SWEET gene with no corresponding naturally occurring TAL effector identified, conferred susceptibility to the Xoo strains expressing the corresponding dTALE genes. Our results demonstrate that dTALEs can be delivered through the bacterial secretion system to activate genes of interest for functional analysis in plants.

  3. Engineering customized TALE nucleases (TALENs) and TALE transcription factors by fast ligation-based automatable solid-phase high-throughput (FLASH) assembly.

    PubMed

    Reyon, Deepak; Maeder, Morgan L; Khayter, Cyd; Tsai, Shengdar Q; Foley, Jonathan E; Sander, Jeffry D; Joung, J Keith

    2013-07-01

    Customized DNA-binding domains made using transcription activator-like effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in various organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or downregulate expression of endogenous genes in human cells and plants. This unit describes a detailed protocol for the recently described fast ligation-based automatable solid-phase high-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multichannel pipet. Using the automated approach, a single researcher can construct up to 96 DNA fragments encoding TALE repeat arrays of various lengths in a single day, and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in a week or less. Plasmids required for FLASH are available by request from the Joung lab (http://eGenome.org). This unit also describes improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) web server (http://ZiFiT.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high throughput.

  4. A TALE nuclease architecture for efficient genome editing.

    PubMed

    Miller, Jeffrey C; Tan, Siyuan; Qiao, Guijuan; Barlow, Kyle A; Wang, Jianbin; Xia, Danny F; Meng, Xiangdong; Paschon, David E; Leung, Elo; Hinkley, Sarah J; Dulay, Gladys P; Hua, Kevin L; Ankoudinova, Irina; Cost, Gregory J; Urnov, Fyodor D; Zhang, H Steve; Holmes, Michael C; Zhang, Lei; Gregory, Philip D; Rebar, Edward J

    2011-02-01

    Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.

  5. DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats

    PubMed Central

    de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

    2015-01-01

    Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. PMID:26481363

  6. Gene knockout using transcription activator-like effector nucleases (TALENs) reveals that human NDUFA9 protein is essential for stabilizing the junction between membrane and matrix arms of complex I.

    PubMed

    Stroud, David A; Formosa, Luke E; Wijeyeratne, Xiaonan W; Nguyen, Thanh N; Ryan, Michael T

    2013-01-18

    Transcription activator-like effector nucleases (TALENs) represent a promising approach for targeted knock-out of genes in cultured human cells. We used TALEN-technology to knock out the nuclear gene encoding NDUFA9, a subunit of mitochondrial respiratory chain complex I in HEK293T cells. Screening for the knock-out revealed a mixture of NDUFA9 cell clones that harbored partial deletions of the mitochondrial N-terminal targeting signal but were still capable of import. A cell line lacking functional copies of both NDUFA9 alleles resulted in a loss of NDUFA9 protein expression, impaired assembly of complex I, and cells incapable of growth in galactose medium. Cells lacking NDUFA9 contained a complex I subcomplex consisting of membrane arm subunits but not marker subunits of the matrix arm. Re-expression of NDUFA9 restored the defects in complex I assembly. We conclude that NDUFA9 is involved in stabilizing the junction between membrane and matrix arms of complex I, a late assembly step critical for complex I biogenesis and activity.

  7. Transcription activator-like effector nuclease (TALEN)-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC) and neural stem cell (NSC) lines.

    PubMed

    Cerbini, Trevor; Funahashi, Ray; Luo, Yongquan; Liu, Chengyu; Park, Kyeyoon; Rao, Mahendra; Malik, Nasir; Zou, Jizhong

    2015-01-01

    Targeted genome engineering to robustly express transgenes is an essential methodology for stem cell-based research and therapy. Although designer nucleases have been used to drastically enhance gene editing efficiency, targeted addition and stable expression of transgenes to date is limited at single gene/locus and mostly PPP1R12C/AAVS1 in human stem cells. Here we constructed transcription activator-like effector nucleases (TALENs) targeting the safe-harbor like gene CLYBL to mediate reporter gene integration at 38%-58% efficiency, and used both AAVS1-TALENs and CLYBL-TALENs to simultaneously knock-in multiple reporter genes at dual safe-harbor loci in human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs). The CLYBL-TALEN engineered cell lines maintained robust reporter expression during self-renewal and differentiation, and revealed that CLYBL targeting resulted in stronger transgene expression and less perturbation on local gene expression than PPP1R12C/AAVS1. TALEN-mediated CLYBL engineering provides improved transgene expression and options for multiple genetic modification in human stem cells.

  8. An Improved Method for TAL Effectors DNA-Binding Sites Prediction Reveals Functional Convergence in TAL Repertoires of Xanthomonas oryzae Strains

    PubMed Central

    Pérez-Quintero, Alvaro L.; Rodriguez-R, Luis M.; Dereeper, Alexis; López, Camilo; Koebnik, Ralf; Szurek, Boris; Cunnac, Sebastien

    2013-01-01

    Transcription Activators-Like Effectors (TALEs) belong to a family of virulence proteins from the Xanthomonas genus of bacterial plant pathogens that are translocated into the plant cell. In the nucleus, TALEs act as transcription factors inducing the expression of susceptibility genes. A code for TALE-DNA binding specificity and high-resolution three-dimensional structures of TALE-DNA complexes were recently reported. Accurate prediction of TAL Effector Binding Elements (EBEs) is essential to elucidate the biological functions of the many sequenced TALEs as well as for robust design of artificial TALE DNA-binding domains in biotechnological applications. In this work a program with improved EBE prediction performances was developed using an updated specificity matrix and a position weight correction function to account for the matching pattern observed in a validation set of TALE-DNA interactions. To gain a systems perspective on the large TALE repertoires from X. oryzae strains, this program was used to predict rice gene targets for 99 sequenced family members. Integrating predictions and available expression data in a TALE-gene network revealed multiple candidate transcriptional targets for many TALEs as well as several possible instances of functional convergence among TALEs. PMID:23869221

  9. Specific DNA-RNA hybrid recognition by TAL effectors.

    PubMed

    Yin, Ping; Deng, Dong; Yan, Chuangye; Pan, Xiaojing; Xi, Jianzhong Jeff; Yan, Nieng; Shi, Yigong

    2012-10-25

    The transcription activator-like (TAL) effector targets specific host promoter through its central DNA-binding domain, which comprises multiple tandem repeats (TALE repeats). Recent structural analyses revealed that the TALE repeats form a superhelical structure that tracks along the forward strand of the DNA duplex. Here, we demonstrate that TALE repeats specifically recognize a DNA-RNA hybrid where the DNA strand determines the binding specificity. The crystal structure of a designed TALE in complex with the DNA-RNA hybrid was determined at a resolution of 2.5 Å. Although TALE repeats are in direct contact with only the DNA strand, the phosphodiester backbone of the RNA strand is inaccessible by macromolecules such as RNases. Consistent with this observation, sequence-specific recognition of an HIV-derived DNA-RNA hybrid by an engineered TALE efficiently blocked RNase H-mediated degradation of the RNA strand. Our study broadens the utility of TALE repeats and suggests potential applications in processes involving DNA replication and retroviral infections.

  10. The Development of TALE Nucleases for Biotechnology.

    PubMed

    Ousterout, David G; Gersbach, Charles A

    2016-01-01

    The development of a facile genome engineering technology based on transcription activator-like effector nucleases (TALENs) has led to significant advances in diverse areas of science and medicine. In this review, we provide a broad overview of the development of TALENs and the use of this technology in basic science, biotechnology, and biomedical applications. This includes the discovery of DNA recognition by TALEs, engineering new TALE proteins to diverse targets, general advances in nuclease-based editing strategies, and challenges that are specific to various applications of the TALEN technology. We review examples of applying TALENs for studying gene function and regulation, generating disease models, and developing gene therapies. The current status of genome editing and future directions for other uses of these technologies are also discussed.

  11. The Development of TALE Nucleases for Biotechnology

    PubMed Central

    Ousterout, David G.; Gersbach, Charles A.

    2017-01-01

    The development of a facile genome engineering technology based on transcription activator-like effector nucleases (TALENs) has led to significant advances in diverse areas of science and medicine. In this review, we provide a broad overview of the development of TALENs and the use of this technology in basic science, biotechnology, and biomedical applications. This includes the discovery of DNA recognition by TALEs, engineering new TALE proteins to diverse targets, general advances in nuclease-based editing strategies, and challenges that are specific to various applications of the TALEN technology. We review examples of applying TALENs for studying gene function and regulation, generating disease models, and developing gene therapies. The current status of genome editing and future directions for other uses of these technologies are also discussed. PMID:26443211

  12. Live visualization of genomic loci with BiFC-TALE

    PubMed Central

    Hu, Huan; Zhang, Hongmin; Wang, Sheng; Ding, Miao; An, Hui; Hou, Yingping; Yang, Xiaojing; Wei, Wensheng; Sun, Yujie; Tang, Chao

    2017-01-01

    Tracking the dynamics of genomic loci is important for understanding the mechanisms of fundamental intracellular processes. However, fluorescent labeling and imaging of such loci in live cells have been challenging. One of the major reasons is the low signal-to-background ratio (SBR) of images mainly caused by the background fluorescence from diffuse full-length fluorescent proteins (FPs) in the living nucleus, hampering the application of live cell genomic labeling methods. Here, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies, we developed a novel method for labeling genomic loci (BiFC-TALE), which largely reduces the background fluorescence level. Using BiFC-TALE, we demonstrated a significantly improved SBR by imaging telomeres and centromeres in living cells in comparison with the methods using full-length FP. PMID:28074901

  13. Context influences on TALE-DNA binding revealed by quantitative profiling.

    PubMed

    Rogers, Julia M; Barrera, Luis A; Reyon, Deepak; Sander, Jeffry D; Kellis, Manolis; Joung, J Keith; Bulyk, Martha L

    2015-06-11

    Transcription activator-like effector (TALE) proteins recognize DNA using a seemingly simple DNA-binding code, which makes them attractive for use in genome engineering technologies that require precise targeting. Although this code is used successfully to design TALEs to target specific sequences, off-target binding has been observed and is difficult to predict. Here we explore TALE-DNA interactions comprehensively by quantitatively assaying the DNA-binding specificities of 21 representative TALEs to ∼5,000-20,000 unique DNA sequences per protein using custom-designed protein-binding microarrays (PBMs). We find that protein context features exert significant influences on binding. Thus, the canonical recognition code does not fully capture the complexity of TALE-DNA binding. We used the PBM data to develop a computational model, Specificity Inference For TAL-Effector Design (SIFTED), to predict the DNA-binding specificity of any TALE. We provide SIFTED as a publicly available web tool that predicts potential genomic off-target sites for improved TALE design.

  14. Dissection of TALE-dependent gene activation reveals that they induce transcription cooperatively and in both orientations

    PubMed Central

    Streubel, Jana; Baum, Heidi; Grau, Jan; Stuttman, Johannes; Boch, Jens

    2017-01-01

    Plant-pathogenic Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators. PMID:28301511

  15. Visualization of specific repetitive genomic sequences with fluorescent TALEs in Arabidopsis thaliana.

    PubMed

    Fujimoto, Satoru; Sugano, Shigeo S; Kuwata, Keiko; Osakabe, Keishi; Matsunaga, Sachihiro

    2016-11-01

    Live imaging of the dynamics of nuclear organization provides the opportunity to uncover the mechanisms responsible for four-dimensional genome architecture. Here, we describe the use of fluorescent protein (FP) fusions of transcription activator-like effectors (TALEs) to visualize endogenous genomic sequences in Arabidopsis thaliana. The ability to engineer sequence-specific TALEs permits the investigation of precise genomic sequences. We could detect TALE-FP signals associated with centromeric, telomeric, and rDNA repeats and the signal distribution was consistent with that observed by fluorescent in situ hybridization. TALE-FPs are advantageous because they permit the observation of intact tissues. We used our TALE-FP method to investigate the nuclei of several multicellular plant tissues including roots, hypocotyls, leaves, and flowers. Because TALE-FPs permit live-cell imaging, we successfully observed the temporal dynamics of centromeres and telomeres in plant organs. Fusing TALEs to multimeric FPs enhanced the signal intensity when observing telomeres. We found that the mobility of telomeres was different in sub-nuclear regions. Transgenic plants stably expressing TALE-FPs will provide new insights into chromatin organization and dynamics in multicellular organisms.

  16. Visualization of specific repetitive genomic sequences with fluorescent TALEs in Arabidopsis thaliana

    PubMed Central

    Fujimoto, Satoru; Sugano, Shigeo S.; Kuwata, Keiko; Osakabe, Keishi; Matsunaga, Sachihiro

    2016-01-01

    Live imaging of the dynamics of nuclear organization provides the opportunity to uncover the mechanisms responsible for four-dimensional genome architecture. Here, we describe the use of fluorescent protein (FP) fusions of transcription activator-like effectors (TALEs) to visualize endogenous genomic sequences in Arabidopsis thaliana. The ability to engineer sequence-specific TALEs permits the investigation of precise genomic sequences. We could detect TALE-FP signals associated with centromeric, telomeric, and rDNA repeats and the signal distribution was consistent with that observed by fluorescent in situ hybridization. TALE-FPs are advantageous because they permit the observation of intact tissues. We used our TALE-FP method to investigate the nuclei of several multicellular plant tissues including roots, hypocotyls, leaves, and flowers. Because TALE-FPs permit live-cell imaging, we successfully observed the temporal dynamics of centromeres and telomeres in plant organs. Fusing TALEs to multimeric FPs enhanced the signal intensity when observing telomeres. We found that the mobility of telomeres was different in sub-nuclear regions. Transgenic plants stably expressing TALE-FPs will provide new insights into chromatin organization and dynamics in multicellular organisms. PMID:27811079

  17. TALE1 from Xanthomonas axonopodis pv. manihotis acts as a transcriptional activator in plant cells and is important for pathogenicity in cassava plants.

    PubMed

    Castiblanco, Luisa F; Gil, Juliana; Rojas, Alejandro; Osorio, Daniela; Gutiérrez, Sonia; Muñoz-Bodnar, Alejandra; Perez-Quintero, Alvaro L; Koebnik, Ralf; Szurek, Boris; López, Camilo; Restrepo, Silvia; Verdier, Valérie; Bernal, Adriana J

    2013-01-01

    Many plant-pathogenic bacteria suppress pathogen-associated molecular pattern (PAMP)-triggered immunity by injecting effector proteins into the host cytoplasm during infection through the type III secretion system (TTSS). This type III secretome plays an important role in bacterial pathogenicity in susceptible hosts. Xanthomonas axonopodis pv. manihotis (Xam), the causal agent of cassava bacterial blight, injects several effector proteins into the host cell, including TALE1(Xam) . This protein is a member of the Transcriptional Activator-Like effector (TALE) protein family, formerly known as the AvrBs3/PthA family. TALE1(Xam) has 13.5 tandem repeats of 34 amino acids each, as well as two nuclear localization signals and an acidic activation domain at the C-terminus. In this work, we demonstrate the importance of TALE1(Xam) in the pathogenicity of Xam. We use versions of the gene that lack different domains in the protein in structure-function studies to show that the eukaryotic domains at the 3' end are critical for pathogenicity. In addition, we demonstrate that, similar to the characterized TALE proteins from other Xanthomonas species, TALE1(Xam) acts as a transcriptional activator in plant cells. This is the first report of the identification of a TALE in Xam, and contributes to our understanding of the pathogenicity mechanisms employed by this bacterium to colonize and cause disease in cassava.

  18. A simple and efficient method for assembling TALE protein based on plasmid library.

    PubMed

    Zhang, Zhiqiang; Li, Duo; Xu, Huarong; Xin, Ying; Zhang, Tingting; Ma, Lixia; Wang, Xin; Chen, Zhilong; Zhang, Zhiying

    2013-01-01

    DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas sp. consists of tandem repeats that can be rearranged according to a simple cipher to target new DNA sequences with high DNA-binding specificity. This technology has been successfully applied in varieties of species for genome engineering. However, assembling long TALE tandem repeats remains a big challenge precluding wide use of this technology. Although several new methodologies for efficiently assembling TALE repeats have been recently reported, all of them require either sophisticated facilities or skilled technicians to carry them out. Here, we described a simple and efficient method for generating customized TALE nucleases (TALENs) and TALE transcription factors (TALE-TFs) based on TALE repeat tetramer library. A tetramer library consisting of 256 tetramers covers all possible combinations of 4 base pairs. A set of unique primers was designed for amplification of these tetramers. PCR products were assembled by one step of digestion/ligation reaction. 12 TALE constructs including 4 TALEN pairs targeted to mouse Gt(ROSA)26Sor gene and mouse Mstn gene sequences as well as 4 TALE-TF constructs targeted to mouse Oct4, c-Myc, Klf4 and Sox2 gene promoter sequences were generated by using our method. The construction routines took 3 days and parallel constructions were available. The rate of positive clones during colony PCR verification was 64% on average. Sequencing results suggested that all TALE constructs were performed with high successful rate. This is a rapid and cost-efficient method using the most common enzymes and facilities with a high success rate.

  19. Genome Engineering with TALE and CRISPR Systems in Neuroscience

    PubMed Central

    Lee, Han B.; Sundberg, Brynn N.; Sigafoos, Ashley N.; Clark, Karl J.

    2016-01-01

    Recent advancement in genome engineering technology is changing the landscape of biological research and providing neuroscientists with an opportunity to develop new methodologies to ask critical research questions. This advancement is highlighted by the increased use of programmable DNA-binding agents (PDBAs) such as transcription activator-like effector (TALE) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems. These PDBAs fused or co-expressed with various effector domains allow precise modification of genomic sequences and gene expression levels. These technologies mirror and extend beyond classic gene targeting methods contributing to the development of novel tools for basic and clinical neuroscience. In this Review, we discuss the recent development in genome engineering and potential applications of this technology in the field of neuroscience. PMID:27092173

  20. Benchmarking of TALE- and CRISPR/dCas9-Based Transcriptional Regulators in Mammalian Cells for the Construction of Synthetic Genetic Circuits.

    PubMed

    Lebar, Tina; Jerala, Roman

    2016-10-21

    Transcriptional activator-like effector (TALE)- and CRISPR/Cas9-based designable recognition domains represent a technological breakthrough not only for genome editing but also for building designed genetic circuits. Both platforms are able to target rarely occurring DNA segments, even within complex genomes. TALE and dCas9 domains, genetically fused to transcriptional regulatory domains, can be used for the construction of engineered logic circuits. Here we benchmarked the performance of the two platforms, targeting the same DNA sequences, to compare their advantages for the construction of designed circuits in mammalian cells. Optimal targeting strands for repression and activation of dCas9-based designed transcription factors were identified; both platforms exhibited good orthogonality and were used to construct functionally complete NOR gates. Although the CRISPR/dCas9 system is clearly easier to construct, TALE-based activators were significantly stronger, and the TALE-based platform performed better, especially for the construction of layered circuits.

  1. Computational predictions provide insights into the biology of TAL effector target sites.

    PubMed

    Grau, Jan; Wolf, Annett; Reschke, Maik; Bonas, Ulla; Posch, Stefan; Boch, Jens

    2013-01-01

    Transcription activator-like (TAL) effectors are injected into host plant cells by Xanthomonas bacteria to function as transcriptional activators for the benefit of the pathogen. The DNA binding domain of TAL effectors is composed of conserved amino acid repeat structures containing repeat-variable diresidues (RVDs) that determine DNA binding specificity. In this paper, we present TALgetter, a new approach for predicting TAL effector target sites based on a statistical model. In contrast to previous approaches, the parameters of TALgetter are estimated from training data computationally. We demonstrate that TALgetter successfully predicts known TAL effector target sites and often yields a greater number of predictions that are consistent with up-regulation in gene expression microarrays than an existing approach, Target Finder of the TALE-NT suite. We study the binding specificities estimated by TALgetter and approve that different RVDs are differently important for transcriptional activation. In subsequent studies, the predictions of TALgetter indicate a previously unreported positional preference of TAL effector target sites relative to the transcription start site. In addition, several TAL effectors are predicted to bind to the TATA-box, which might constitute one general mode of transcriptional activation by TAL effectors. Scrutinizing the predicted target sites of TALgetter, we propose several novel TAL effector virulence targets in rice and sweet orange. TAL-mediated induction of the candidates is supported by gene expression microarrays. Validity of these targets is also supported by functional analogy to known TAL effector targets, by an over-representation of TAL effector targets with similar function, or by a biological function related to pathogen infection. Hence, these predicted TAL effector virulence targets are promising candidates for studying the virulence function of TAL effectors. TALgetter is implemented as part of the open-source Java library

  2. Libraries of Synthetic TALE-Activated Promoters: Methods and Applications.

    PubMed

    Schreiber, T; Tissier, A

    2016-01-01

    The discovery of proteins with programmable DNA-binding specificities triggered a whole array of applications in synthetic biology, including genome editing, regulation of transcription, and epigenetic modifications. Among those, transcription activator-like effectors (TALEs) due to their natural function as transcription regulators, are especially well-suited for the development of orthogonal systems for the control of gene expression. We describe here the construction and testing of libraries of synthetic TALE-activated promoters which are under the control of a single TALE with a given DNA-binding specificity. These libraries consist of a fixed DNA-binding element for the TALE, a TATA box, and variable sequences of 19 bases upstream and 43 bases downstream of the DNA-binding element. These libraries were cloned using a Golden Gate cloning strategy making them usable as standard parts in a modular cloning system. The broad range of promoter activities detected and the versatility of these promoter libraries make them valuable tools for applications in the fine-tuning of expression in metabolic engineering projects or in the design and implementation of regulatory circuits.

  3. A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets

    PubMed Central

    Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

    2014-01-01

    Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways. PMID:25475013

  4. A host basal transcription factor is a key component for infection of rice by TALE-carrying bacteria

    PubMed Central

    Yuan, Meng; Ke, Yinggen; Huang, Renyan; Ma, Ling; Yang, Zeyu; Chu, Zhaohui; Xiao, Jinghua; Li, Xianghua; Wang, Shiping

    2016-01-01

    Transcription activator-like effectors (TALEs) are sequence-specific DNA binding proteins found in a range of plant pathogenic bacteria, where they play important roles in host-pathogen interactions. However, it has been unclear how TALEs, after they have been injected into the host cells, activate transcription of host genes required for infection success. Here, we show that the basal transcription factor IIA gamma subunit TFIIAγ5 from rice is a key component for infection by the TALE-carrying bacterium Xanthomonas oryzae pv. oryzae, the causal agent for bacterial blight. Direct interaction of several TALEs with TFIIAγ5 is required for activation of disease susceptibility genes. Conversely, reduced expression of the TFIIAγ5 host gene limits the induction of susceptibility genes and thus decreases bacterial blight symptoms. Suppression or mutation of TFIIAγ5 can also reduce bacterial streak, another devastating disease of rice caused by TALE-carrying X. oryzae pv. oryzicola. These results have important implications for formulating a widely applicable strategy with which to improve resistance of plants to TALE-carrying pathogens. DOI: http://dx.doi.org/10.7554/eLife.19605.001 PMID:27472897

  5. A host basal transcription factor is a key component for infection of rice by TALE-carrying bacteria.

    PubMed

    Yuan, Meng; Ke, Yinggen; Huang, Renyan; Ma, Ling; Yang, Zeyu; Chu, Zhaohui; Xiao, Jinghua; Li, Xianghua; Wang, Shiping

    2016-07-29

    Transcription activator-like effectors (TALEs) are sequence-specific DNA binding proteins found in a range of plant pathogenic bacteria, where they play important roles in host-pathogen interactions. However, it has been unclear how TALEs, after they have been injected into the host cells, activate transcription of host genes required for infection success. Here, we show that the basal transcription factor IIA gamma subunit TFIIAγ5 from rice is a key component for infection by the TALE-carrying bacterium Xanthomonas oryzae pv. oryzae, the causal agent for bacterial blight. Direct interaction of several TALEs with TFIIAγ5 is required for activation of disease susceptibility genes. Conversely, reduced expression of the TFIIAγ5 host gene limits the induction of susceptibility genes and thus decreases bacterial blight symptoms. Suppression or mutation of TFIIAγ5 can also reduce bacterial streak, another devastating disease of rice caused by TALE-carrying X. oryzae pv. oryzicola. These results have important implications for formulating a widely applicable strategy with which to improve resistance of plants to TALE-carrying pathogens.

  6. Suppression of Xo1-Mediated Disease Resistance in Rice by a Truncated, Non-DNA-Binding TAL Effector of Xanthomonas oryzae

    PubMed Central

    Read, Andrew C.; Rinaldi, Fabio C.; Hutin, Mathilde; He, Yong-Qiang; Triplett, Lindsay R.; Bogdanove, Adam J.

    2016-01-01

    Delivered into plant cells by type III secretion from pathogenic Xanthomonas species, TAL (transcription activator-like) effectors are nuclear-localized, DNA-binding proteins that directly activate specific host genes. Targets include genes important for disease, genes that confer resistance, and genes inconsequential to the host-pathogen interaction. TAL effector specificity is encoded by polymorphic repeats of 33–35 amino acids that interact one-to-one with nucleotides in the recognition site. Activity depends also on N-terminal sequences important for DNA binding and C-terminal nuclear localization signals (NLS) and an acidic activation domain (AD). Coding sequences missing much of the N- and C-terminal regions due to conserved, in-frame deletions are present and annotated as pseudogenes in sequenced strains of Xanthomonas oryzae pv. oryzicola (Xoc) and pv. oryzae (Xoo), which cause bacterial leaf streak and bacterial blight of rice, respectively. Here we provide evidence that these sequences encode proteins we call “truncTALEs,” for “truncated TAL effectors.” We show that truncTALE Tal2h of Xoc strain BLS256, and by correlation truncTALEs in other strains, specifically suppress resistance mediated by the Xo1 locus recently described in the heirloom rice variety Carolina Gold. Xo1-mediated resistance is triggered by different TAL effectors from diverse X. oryzae strains, irrespective of their DNA binding specificity, and does not require the AD. This implies a direct protein-protein rather than protein-DNA interaction. Similarly, truncTALEs exhibit diverse predicted DNA recognition specificities. And, in vitro, Tal2h did not bind any of several potential recognition sites. Further, a single candidate NLS sequence in Tal2h was dispensable for resistance suppression. Many truncTALEs have one 28 aa repeat, a length not observed previously. Tested in an engineered TAL effector, this repeat required a single base pair deletion in the DNA, suggesting that it

  7. Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease.

    PubMed

    de Lange, Orlando; Schreiber, Tom; Schandry, Niklas; Radeck, Jara; Braun, Karl Heinz; Koszinowski, Julia; Heuer, Holger; Strauß, Annett; Lahaye, Thomas

    2013-08-01

    Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We have characterized Brg11(hrpB-regulated 11), the first identified member of a class of Rips with high sequence similarity to the transcription activator-like (TAL) effectors of Xanthomonas spp., collectively termed RipTALs. Fluorescence microscopy of in planta expressed RipTALs showed nuclear localization. Domain swaps between Brg11 and Xanthomonas TAL effector (TALE) AvrBs3 (avirulence protein triggering Bs3 resistance) showed the functional interchangeability of DNA-binding and transcriptional activation domains. PCR was used to determine the sequence of brg11 homologs from strains infecting phylogenetically diverse host plants. Brg11 localizes to the nucleus and activates promoters containing a matching effector-binding element (EBE). Brg11 and homologs preferentially activate promoters containing EBEs with a 5' terminal guanine, contrasting with the TALE preference for a 5' thymine. Brg11 and other RipTALs probably promote disease through the transcriptional activation of host genes. Brg11 and the majority of homologs identified in this study were shown to activate similar or identical target sequences, in contrast to TALEs, which generally show highly diverse target preferences. This information provides new options for the engineering of plants resistant to R. solanacearum.

  8. RNA-seq pinpoints a Xanthomonas TAL-effector activated resistance gene in a large-crop genome.

    PubMed

    Strauss, Tina; van Poecke, Remco M P; Strauss, Annett; Römer, Patrick; Minsavage, Gerald V; Singh, Sylvia; Wolf, Christina; Strauss, Axel; Kim, Seungill; Lee, Hyun-Ah; Yeom, Seon-In; Parniske, Martin; Stall, Robert E; Jones, Jeffrey B; Choi, Doil; Prins, Marcel; Lahaye, Thomas

    2012-11-20

    Transcription activator-like effector (TALE) proteins of the plant pathogenic bacterial genus Xanthomonas bind to and transcriptionally activate host susceptibility genes, promoting disease. Plant immune systems have taken advantage of this mechanism by evolving TALE binding sites upstream of resistance (R) genes. For example, the pepper Bs3 and rice Xa27 genes are hypersensitive reaction plant R genes that are transcriptionally activated by corresponding TALEs. Both R genes have a hallmark expression pattern in which their transcripts are detectable only in the presence and not the absence of the corresponding TALE. By transcriptome profiling using next-generation sequencing (RNA-seq), we tested whether we could avoid laborious positional cloning for the isolation of TALE-induced R genes. In a proof-of-principle experiment, RNA-seq was used to identify a candidate for Bs4C, an R gene from pepper that mediates recognition of the Xanthomonas TALE protein AvrBs4. We identified one major Bs4C candidate transcript by RNA-seq that was expressed exclusively in the presence of AvrBs4. Complementation studies confirmed that the candidate corresponds to the Bs4C gene and that an AvrBs4 binding site in the Bs4C promoter directs its transcriptional activation. Comparison of Bs4C with a nonfunctional allele that is unable to recognize AvrBs4 revealed a 2-bp polymorphism within the TALE binding site of the Bs4C promoter. Bs4C encodes a structurally unique R protein and Bs4C-like genes that are present in many solanaceous genomes seem to be as tightly regulated as pepper Bs4C. These findings demonstrate that TALE-specific R genes can be cloned from large-genome crops with a highly efficient RNA-seq approach.

  9. Spatial organization of heterologous metabolic system in vivo based on TALE

    PubMed Central

    Zhu, Ling-yun; Qiu, Xin-yuan; Zhu, Ling-yun; Wu, Xiao-min; Zhang, Yuan; Zhu, Qian-hui; Fan, Dong-yu; Zhu, Chu-shu; Zhang, Dong-yi

    2016-01-01

    For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR. The effect of the system was determined through a split GFP study and validated through the heterologous production of indole-3-acetic acid (IAA) by incorporating TALE-fused IAA biosynthetic enzymes in E. coli. To the best of our knowledge, we are the first to use the TALE system as a scaffold for the spatial organization of bacterial metabolism. This technique might be used to establish multi-enzymatic reaction programs in a prokaryotic chassis for various applications. PMID:27184291

  10. Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands.

    PubMed

    Luo, Yan; Wang, Yongsheng; Liu, Jun; Cui, Chenchen; Wu, Yongyan; Lan, Hui; Chen, Qi; Liu, Xu; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2016-02-08

    Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription-activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.

  11. Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands

    PubMed Central

    Luo, Yan; Wang, Yongsheng; Liu, Jun; Cui, Chenchen; Wu, Yongyan; Lan, Hui; Chen, Qi; Liu, Xu; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2016-01-01

    Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription- activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock. PMID:26853907

  12. Creating a monomeric endonuclease TALE-I-SceI with high specificity and low genotoxicity in human cells

    PubMed Central

    Lin, Jianfei; Chen, He; Luo, Ling; Lai, Yongrong; Xie, Wei; Kee, Kehkooi

    2015-01-01

    To correct a DNA mutation in the human genome for gene therapy, homology-directed repair (HDR) needs to be specific and have the lowest off-target effects to protect the human genome from deleterious mutations. Zinc finger nucleases, transcription activator-like effector nuclease (TALEN) and CRISPR-CAS9 systems have been engineered and used extensively to recognize and modify specific DNA sequences. Although TALEN and CRISPR/CAS9 could induce high levels of HDR in human cells, their genotoxicity was significantly higher. Here, we report the creation of a monomeric endonuclease that can recognize at least 33 bp by fusing the DNA-recognizing domain of TALEN (TALE) to a re-engineered homing endonuclease I-SceI. After sequentially re-engineering I-SceI to recognize 18 bp of the human β-globin sequence, the re-engineered I-SceI induced HDR in human cells. When the re-engineered I-SceI was fused to TALE (TALE-ISVB2), the chimeric endonuclease induced the same HDR rate at the human β-globin gene locus as that induced by TALEN, but significantly reduced genotoxicity. We further demonstrated that TALE-ISVB2 specifically targeted at the β-globin sequence in human hematopoietic stem cells. Therefore, this monomeric endonuclease has the potential to be used in therapeutic gene targeting in human cells. PMID:25541197

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

    PubMed

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

    2014-04-01

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

  14. Fairy Tale Autobiographies.

    ERIC Educational Resources Information Center

    Schulze, Patricia

    Students will work in groups to read and analyze fairy tales, brainstorm for events in their lives that could be changed into fairy tales, develop setting, characters, and plot for their fairy tale, write, illustrate, and compile their fairy tales into group books. During ten 45-minute lessons, students will: choose three fairy tales and read them…

  15. [Tale nucleases--new tool for genome editing].

    PubMed

    Glazkova, D V; Shipulin, G A

    2014-01-01

    The ability to introduce targeted changes in the genome of living cells or entire organisms enables researchers to meet the challenges of basic life sciences, biotechnology and medicine. Knockdown of target genes in the zygotes gives the opportunity to investigate the functions of these genes in different organisms. Replacement of single nucleotide in the DNA sequence allows to correct mutations in genes and thus to cure hereditary diseases. Adding transgene to specific genomic.loci can be used in biotechnology for generation of organisms with certain properties or cell lines for biopharmaceutical production. Such manipulations of gene sequences in their natural chromosomal context became possible after the emergence of the technology called "genome editing". This technology is based on the induction of a double-strand break in a specific genomic target DNA using endonucleases that recognize the unique sequences in the genome and on subsequent recovery of DNA integrity through the use of cellular repair mechanisms. A necessary tool for the genome editing is a custom-designed endonuclease which is able to recognize selected sequences. The emergence of a new type of programmable endonucleases, which were constructed on the basis of bacterial proteins--TAL-effectors (Transcription activators like effector), has become an important stage in the development of technology and promoted wide spread of the genome editing. This article reviews the history of the discovery of TAL effectors and creation of TALE nucleases, and describes their advantages over zinc finger endonucleases that appeared earlier. A large section is devoted to description of genetic modifications that can be performed using the genome editing.

  16. A TALE of Transposition: Tn3-Like Transposons Play a Major Role in the Spread of Pathogenicity Determinants of Xanthomonas citri and Other Xanthomonads

    PubMed Central

    Ferreira, Rafael Marini; de Oliveira, Amanda Carolina P.; Moreira, Leandro M.; Belasque, José; Gourbeyre, Edith; Siguier, Patricia; Ferro, Maria Inês T.; Ferro, Jesus A.

    2015-01-01

    ABSTRACT Members of the genus Xanthomonas are among the most important phytopathogens. A key feature of Xanthomonas pathogenesis is the translocation of type III secretion system (T3SS) effector proteins (T3SEs) into the plant target cells via a T3SS. Several T3SEs and a murein lytic transglycosylase gene (mlt, required for citrus canker symptoms) are found associated with three transposition-related genes in Xanthomonas citri plasmid pXAC64. These are flanked by short inverted repeats (IRs). The region was identified as a transposon, TnXax1, with typical Tn3 family features, including a transposase and two recombination genes. Two 14-bp palindromic sequences within a 193-bp potential resolution site occur between the recombination genes. Additional derivatives carrying different T3SEs and other passenger genes occur in different Xanthomonas species. The T3SEs include transcription activator-like effectors (TALEs). Certain TALEs are flanked by the same IRs as found in TnXax1 to form mobile insertion cassettes (MICs), suggesting that they may be transmitted horizontally. A significant number of MICs carrying other passenger genes (including a number of TALE genes) were also identified, flanked by the same TnXax1 IRs and delimited by 5-bp target site duplications. We conclude that a large fraction of T3SEs, including individual TALEs and potential pathogenicity determinants, have spread by transposition and that TnXax1, which exhibits all of the essential characteristics of a functional transposon, may be involved in driving MIC transposition. We also propose that TALE genes may diversify by fork slippage during the replicative Tn3 family transposition. These mechanisms may play a crucial role in the emergence of Xanthomonas pathogenicity. PMID:25691597

  17. An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes

    PubMed Central

    Barbon, Elena; Pignani, Silvia; Branchini, Alessio; Bernardi, Francesco; Pinotti, Mirko; Bovolenta, Matteo

    2016-01-01

    Tailored approaches to restore defective transcription responsible for severe diseases have been poorly explored. We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model. In this model, the deficiency is caused by the −94C > G or −61T > G mutation, which abrogate the binding of Sp1 or HNF-4 transcription factors. Reporter assays in hepatoma HepG2 cells naturally expressing FVII identified a single TALE-TF (TF4) that, by targeting the region between mutations, specifically trans-activated both the variant (>100-fold) and wild-type (20–40-fold) F7 promoters. Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene. The ectopic F7 expression in renal HEK293 cells was modestly affected by TF4 or by TALE-TF combinations. These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations. PMID:27341548

  18. Single-cell analyses of regulatory network perturbations using enhancer-targeting TALEs suggest novel roles for PU.1 during haematopoietic specification

    PubMed Central

    Wilkinson, Adam C.; Kawata, Viviane K. S.; Schütte, Judith; Gao, Xuefei; Antoniou, Stella; Baumann, Claudia; Woodhouse, Steven; Hannah, Rebecca; Tanaka, Yosuke; Swiers, Gemma; Moignard, Victoria; Fisher, Jasmin; Hidetoshi, Shimauchi; Tijssen, Marloes R.; de Bruijn, Marella F. T. R.; Liu, Pentao; Göttgens, Berthold

    2014-01-01

    Transcription factors (TFs) act within wider regulatory networks to control cell identity and fate. Numerous TFs, including Scl (Tal1) and PU.1 (Spi1), are known regulators of developmental and adult haematopoiesis, but how they act within wider TF networks is still poorly understood. Transcription activator-like effectors (TALEs) are a novel class of genetic tool based on the modular DNA-binding domains of Xanthomonas TAL proteins, which enable DNA sequence-specific targeting and the manipulation of endogenous gene expression. Here, we report TALEs engineered to target the PU.1-14kb and Scl+40kb transcriptional enhancers as efficient new tools to perturb the expression of these key haematopoietic TFs. We confirmed the efficiency of these TALEs at the single-cell level using high-throughput RT-qPCR, which also allowed us to assess the consequences of both PU.1 activation and repression on wider TF networks during developmental haematopoiesis. Combined with comprehensive cellular assays, these experiments uncovered novel roles for PU.1 during early haematopoietic specification. Finally, transgenic mouse studies confirmed that the PU.1-14kb element is active at sites of definitive haematopoiesis in vivo and PU.1 is detectable in haemogenic endothelium and early committing blood cells. We therefore establish TALEs as powerful new tools to study the functionality of transcriptional networks that control developmental processes such as early haematopoiesis. PMID:25252941

  19. Richard Halliburton's Bearded Tales

    ERIC Educational Resources Information Center

    Morris, Charles E., III

    2009-01-01

    Fusing the concept of "the beard" with the genre of the tall tale to theorize bearded tales deepens our understanding of closet eloquence, or rhetorical repertories of sexual passing in U.S. history. An examination of adventurer-writer-lecturer Richard Halliburton's sexual provenance and bestselling travel tale, "The Royal Road to Romance" (1925),…

  20. Fairy Tales from Life.

    ERIC Educational Resources Information Center

    Schulze, Patricia

    With the help of the teacher, students will read fairy tales and identify common elements. Choosing common situations and working in small groups, students will draw storyboards of their fairy tale and then write the fairy tale. Project will conclude with class presentations. During ten 50-minute lessons, grade 3-5 students will: listen and read…

  1. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers

    PubMed Central

    Briggs, Adrian W.; Rios, Xavier; Chari, Raj; Yang, Luhan; Zhang, Feng; Mali, Prashant; Church, George M.

    2012-01-01

    DNA built from modular repeats presents a challenge for gene synthesis. We present a solid surface-based sequential ligation approach, which we refer to as iterative capped assembly (ICA), that adds DNA repeat monomers individually to a growing chain while using hairpin ‘capping’ oligonucleotides to block incompletely extended chains, greatly increasing the frequency of full-length final products. Applying ICA to a model problem, construction of custom transcription activator-like effector nucleases (TALENs) for genome engineering, we demonstrate efficient synthesis of TALE DNA-binding domains up to 21 monomers long and their ligation into a nuclease-carrying backbone vector all within 3 h. We used ICA to synthesize 20 TALENs of varying DNA target site length and tested their ability to stimulate gene editing by a donor oligonucleotide in human cells. All the TALENS show activity, with the ones >15 monomers long tending to work best. Since ICA builds full-length constructs from individual monomers rather than large exhaustive libraries of pre-fabricated oligomers, it will be trivial to incorporate future modified TALE monomers with improved or expanded function or to synthesize other types of repeat-modular DNA where the diversity of possible monomers makes exhaustive oligomer libraries impractical. PMID:22740649

  2. TALE-induced bHLH transcription factors that activate a pectate lyase contribute to water soaking in bacterial spot of tomato

    PubMed Central

    Schwartz, Allison R.; Morbitzer, Robert; Lahaye, Thomas; Staskawicz, Brian J.

    2017-01-01

    AvrHah1 [avirulence (avr) gene homologous to avrBs3 and hax2, no. 1] is a transcription activator-like (TAL) effector (TALE) in Xanthomonas gardneri that induces water-soaked disease lesions on fruits and leaves during bacterial spot of tomato. We observe that water from outside the leaf is drawn into the apoplast in X. gardneri-infected, but not X. gardneriΔavrHah1 (XgΔavrHah1)-infected, plants, conferring a dark, water-soaked appearance. The pull of water can facilitate entry of additional bacterial cells into the apoplast. Comparing the transcriptomes of tomato infected with X. gardneri vs. XgΔavrHah1 revealed the differential up-regulation of two basic helix–loop–helix (bHLH) transcription factors with predicted effector binding elements (EBEs) for AvrHah1. We mined our RNA-sequencing data for differentially up-regulated genes that could be direct targets of the bHLH transcription factors and therefore indirect targets of AvrHah1. We show that two pectin modification genes, a pectate lyase and pectinesterase, are targets of both bHLH transcription factors. Designer TALEs (dTALEs) for the bHLH transcription factors and the pectate lyase, but not for the pectinesterase, complement water soaking when delivered by XgΔavrHah1. By perturbing transcriptional networks and/or modifying the plant cell wall, AvrHah1 may promote water uptake to enhance tissue damage and eventual bacterial egression from the apoplast to the leaf surface. Understanding how disease symptoms develop may be a useful tool for improving the tolerance of crops from damaging disease lesions. PMID:28100489

  3. Tall Tales on Tour.

    ERIC Educational Resources Information Center

    Renaud, Linda; Silk, Lynn

    "Tall Tales on Tour" consists of a mobile unit of books, posters, puppets, plays, videos, teachers' guides and other resources designed to kindle enthusiasm and love for tall tales in students in grades 3-8 and to establish links with rural Washington County, Maine, schools. The program consists of three main components: (1) immersing…

  4. Tales From Silver Lands.

    ERIC Educational Resources Information Center

    Finger, Charles J.

    In 1925, "Tales From Silver Lands" was awarded the Newbery medal as the most distinguished contribution to American children's literature for the year. The book contains a collection of 19 short stories learned from the Indians of South America as the author traveled to different lands. As described on the dust jacket, the tales are…

  5. Bulgarians: Four Folk Tales.

    ERIC Educational Resources Information Center

    Parpulova, Lubomira

    The four Bulgarian folk tales presented in this booklet are part of an ethnic heritage studies teaching unit on Bulgarian culture. The objective of the cultural awareness project is to help American students in elementary, junior high, and high school understand and appreciate Bulgarians and their culture. Titles of the folk tales are "Poor…

  6. Lost Opportunities: Rediscovering Fairy Tales

    ERIC Educational Resources Information Center

    Wipf, Joan Brogan; Da Ros-Voseles, Denise

    2012-01-01

    The power of fairy tales resonates with children around the world. Fairy tales connect children on an emotional level that can help guide them through the complexities of everyday life. The tales provide stories rich in cultural heritage and the human condition, stories that not only delight children but also instruct. Because fairy tales state…

  7. TAL effectors and the executor R genes

    PubMed Central

    Zhang, Junli; Yin, Zhongchao; White, Frank

    2015-01-01

    Transcription activator-like (TAL) effectors are bacterial type III secretion proteins that function as transcription factors in plants during Xanthomonas/plant interactions, conditioning either host susceptibility and/or host resistance. Three types of TAL effector associated resistance (R) genes have been characterized—recessive, dominant non-transcriptional, and dominant TAL effector-dependent transcriptional based resistance. Here, we discuss the last type of R genes, whose functions are dependent on direct TAL effector binding to discrete effector binding elements in the promoters. Only five of the so-called executor R genes have been cloned, and commonalities are not clear. We have placed the protein products in two groups for conceptual purposes. Group 1 consists solely of the protein from pepper, BS3, which is predicted to have catalytic function on the basis of homology to a large conserved protein family. Group 2 consists of BS4C-R, XA27, XA10, and XA23, all of which are relatively short proteins from pepper or rice with multiple potential transmembrane domains. Group 2 members have low sequence similarity to proteins of unknown function in closely related species. Firm predictions await further experimentation on these interesting new members to the R gene repertoire, which have potential broad application in new strategies for disease resistance. PMID:26347759

  8. Tales of Accreditation Woe.

    ERIC Educational Resources Information Center

    Dickmeyer, Nathan

    2002-01-01

    Offers cautionary tales depicting how an "Enron mentality" infiltrated three universities and jeopardized their accreditation status. The schools were guilty, respectively, of bad bookkeeping, lack of strategy and stable leadership, and loss of academic integrity by selling degrees. (EV)

  9. Translocated effectors of Yersinia

    PubMed Central

    Matsumoto, Hiroyuki; Young, Glenn M.

    2009-01-01

    Summary Currently, all known translocated effectors of Yersinia are delivered into host cells by type III secretion systems (T3SSs). Pathogenic Yersinia maintain the plasmid-encoded Ysc T3SS for the specific delivery of the well-studied Yop effectors. New horizons for effector biology have opened with the discovery of the Ysps of Y. enterocolitica Biovar 1B, which are translocated into host cells by the chromosome-endoded Ysa T3SS. The reported arsenal of effectors is likely to expand since genomic analysis has revealed gene-clusters in some Yersinia that code for other T3SSs. These efforts also revealed possible type VI secretion (T6S) systems, which may indicate translocation of effectors occurs by multiple mechanisms. PMID:19185531

  10. TAL effector-mediated susceptibility to bacterial blight of cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial blight of cotton (BBC) caused by Xanthomonas campestris pv. malvacearum (Xcm) is a destructive disease that has recently re-emerged in the U.S. Xcm injects transcription activator-like (TAL) effectors that directly induce the expression of host susceptibility (S) or resistance (R) genes. ...

  11. Twisters, Tall Tales, & Science Teaching

    ERIC Educational Resources Information Center

    Wilcox, Dawn Renee; Sterling, Donna R.

    2006-01-01

    Legends and tall tales have been part of the American culture for ages. Students are probably already familiar with the tales of how Pecos Bill fearlessly tamed a ferocious tornado, or Paul Bunyan effortlessly restrained a great river. Such tales have been passed down from generation to generation to explain humanity, the natural world, and…

  12. Advanced Aerodynamic Control Effectors

    NASA Technical Reports Server (NTRS)

    Wood, Richard M.; Bauer, Steven X. S.

    1999-01-01

    A 1990 research program that focused on the development of advanced aerodynamic control effectors (AACE) for military aircraft has been reviewed and summarized. Data are presented for advanced planform, flow control, and surface contouring technologies. The data show significant increases in lift, reductions in drag, and increased control power, compared to typical aerodynamic designs. The results presented also highlighted the importance of planform selection in the design of a control effector suite. Planform data showed that dramatic increases in lift (greater than 25%) can be achieved with multiple wings and a sawtooth forebody. Passive porosity and micro drag generator control effector data showed control power levels exceeding that available from typical effectors (moving surfaces). Application of an advanced planform to a tailless concept showed benefits of similar magnitude as those observed in the generic studies.

  13. End-effector microprocessor

    NASA Technical Reports Server (NTRS)

    Doggett, William R.

    1992-01-01

    The topics are presented in viewgraph form and include: automated structures assembly facility current control hierarchy; automated structures assembly facility purposed control hierarchy; end-effector software state transition diagram; block diagram for ideal install composite; and conclusions.

  14. [TALE nuclease engineering and targeted genome modification].

    PubMed

    Shen, Yan; Xiao, An; Huang, Peng; Wang, Wei-Ye; Zhu, Zuo-Yan; Zhang, Bo

    2013-04-01

    Artificial designer nucleases targeting specific DNA sequences open up a new field for reverse genetics study. The rapid development of engineered endonucleases (EENs) enables targeted genome modification theoretically in any species. The construction of transcription activator-like effector nucleases (TALENs) is simpler with higher specificity and less toxicity than zinc-finger nucleases (ZFNs). Here, we summarized the recent progresses and prospects of TALEN technology, with an emphasis on its structure, function, and construction strategies, as well as a collection of species and genes that have been successfully modified by TALENs, especially the application in zebrafish.

  15. TALE nucleases: tailored genome engineering made easy.

    PubMed

    Mussolino, Claudio; Cathomen, Toni

    2012-10-01

    Custom-made designer nucleases have evolved into an indispensable platform to precisely alter complex genomes for basic research, biotechnology, synthetic biology, or human gene therapy. In this review we describe how transcription activator-like effector nucleases (TALENs) have rapidly developed into a chief technology for targeted genome editing in different model organisms as well as human stem cells. We summarize the technological background and provide an overview of the current state-of-the-art of TALENs with regard to activity and specificity of these nucleases for targeted genome engineering.

  16. A Wichita Migration Tale

    ERIC Educational Resources Information Center

    John, Elizabeth

    1983-01-01

    Captured in extraordinary detail in the early 19th century, when Wichita elders then living on the Red River could remember their birthplace on the Arkansas River, the tale reflects the anguish of a people fleeing for their lives, on foot, down the treeless grasslands of the Great Plains. (Author).

  17. Artificial TALE as a Convenient Protein Platform for Engineering Broad-Spectrum Resistance to Begomoviruses

    PubMed Central

    Cheng, Xiaofei; Li, Fangfang; Cai, Jianyu; Chen, Wei; Zhao, Nan; Sun, Yuqiang; Guo, Yushuang; Yang, Xiuling; Wu, Xiaoyun

    2015-01-01

    Transcription activator–like effectors (TALEs) are a class of sequence-specific DNA-binding proteins that utilize a simple and predictable modality to recognize target DNA. This unique characteristic allows for the rapid assembly of artificial TALEs, with high DNA binding specificity, to any target DNA sequences for the creation of customizable sequence-specific nucleases used in genome engineering. Here, we report the use of an artificial TALE protein as a convenient platform for designing broad-spectrum resistance to begomoviruses, one of the most destructive plant virus groups, which cause tremendous losses worldwide. We showed that artificial TALEs, which were assembled based on conserved sequence motifs within begomovirus genomes, could confer partial resistance in transgenic Nicotiana benthamiana to all three begomoviruses tested. Furthermore, the resistance was maintained even in the presence of their betasatellite. These results shed new light on the development of broad-spectrum resistance against DNA viruses, such as begomoviruses. PMID:26308041

  18. Pneumatic inflatable end effector

    NASA Technical Reports Server (NTRS)

    Clark, K. H.; Johnston, J. D. (Inventor)

    1981-01-01

    The invention relates to an end effector device for robot or teleoperated type space vehicle which includes an inflatable balloon member carried on the end of tubular member which has a hollow center or conduit through which a suitable pressurized fluid is supplied. The device may be inserted into a variety of shaped openings or truss-type structures for handling in space.

  19. Designing and testing the activities of TAL effector nucleases.

    PubMed

    Lin, Yanni; Cradick, Thomas J; Bao, Gang

    2014-01-01

    Transcription activator-like effector nucleases (TALENs) have rapidly developed into a powerful tool for genome editing. To avoid labor-intensive and time-consuming experimental screening for active TALENs, a scoring system can help select optimal target sites. Here we describe a procedure to design active TALENs using a scoring system named Scoring Algorithm for Predicted TALEN Activity (SAPTA) and a method to test the activity of individual and pairs of TALENs.

  20. Robotic end effector

    DOEpatents

    Minichan, R.L.

    1993-10-05

    An end effector is described for use in probing a surface with a robotic arm. The end effector has a first portion that carries a gimbal with a probe, the gimbal holding the probe normal to the surface, and a second portion with a set of three shafts within a housing for urging the gimbal and probe against the surface. The second portion contains a potentiometer connected by another shaft to the first portion to measure the position of the first portion with respect to the second so that the second portion can be moved to place and maintain the shafts at the midpoint of their travel. Then, as irregularities in the surface are encountered, the first portion can respond by moving closer to or farther from the second portion. 7 figures.

  1. Robotic end effector

    DOEpatents

    Minichan, Richard L.

    1993-01-01

    An end effector for use in probing a surface with a robotic arm. The end effector has a first portion that carries a gimbal with a probe, the gimbal holding the probe normal to the surface, and a second portion with a set of three shafts within a housing for urging the gimbal and probe against the surface. The second portion contains a potentiometer connected by another shaft to the first portion to measure the position of the first portion with respect to the second so that the second portion can be moved to place and maintain the shafts at the midpoint of their travel. Then, as irregularities in the surface are encountered, the first portion can respond by moving closer to or farther from the second portion.

  2. Robotic end effector

    SciTech Connect

    Minichan, R.L.

    1991-12-31

    This invention is comprised of an end effector for use in probing a surface with a robotic arm. The end effector has a first portion that carries a gamble with a probe, the gamble holding the probe normal to the surface, and a second portion with a set of three shafts within a housing for urging the gamble and probe against the surface. The second portion contains a potentiometer connected by another shaft to the first portion to measure the position of the first portion with respect to the second so that the second portion can be moved to place and maintain the shafts at the midpoint of their travel. Then, as irregularities in the surface are encountered, the first portion can respond by moving closer to or farther from the second portion.

  3. Tall Tales of North America.

    ERIC Educational Resources Information Center

    Fresno City Unified School District, CA.

    Designed for use in junior high school language arts classes, this learning activity packet introduces students to North American folklore. Selected readings cover Indian tales, real folk heroes (Davy Crockett and John Henry), imaginary folk heroes (Paul Bunyan and Pecos Bill), Black folk stories (Brer Rabbit), and tales of Washington Irving. Each…

  4. Reading: Tales for Two Actors.

    ERIC Educational Resources Information Center

    Slattery, Judith M.

    1979-01-01

    The author explains how she changes folk tales into simple two-actor plays geared to the oral reading ability of her remedial reading students. Two of her plays are included in this article: "A Halloween Tale" and "The Three Wishes." (SJL)

  5. What Is a Folk Tale?

    ERIC Educational Resources Information Center

    Barnet, Judith M.

    1978-01-01

    Suggests how social studies classroom teachers can help students understand culture and folk art through study of folk tales. Activities involve students in class and group discussions of other cultures, story telling, illustrating folk tales, playing traditional games, and studying traditional foods and medicines. (Author/DB)

  6. Yee-e-e-Haw!: Tall Tales.

    ERIC Educational Resources Information Center

    Jordan, Anne Devereaux

    1997-01-01

    Outlines the characteristics of "tall tales." Fills in the historical background of tall tales, from the ancient myth of Gilgamesh to Baron Munchausen, the closest European progenitor of American tall tales. Opines that tall tales appear to have been created as a response to challenges posed by building a new nation. Lists 11…

  7. Deconstructing Gender in Revised Feminist Fairy Tales

    ERIC Educational Resources Information Center

    Mcandrew, Linda

    2013-01-01

    Power relationships are a central premise in children's literature, especially traditional fairy tales and modern feminist fairy tales. This is seen in many fairy tales where the main female character is in some distress, her Prince Charming rescues her, and they live happily ever after. Modern feminist fairy tales are understood to be a forum…

  8. Direct regulation of E-cadherin by targeted histone methylation of TALE-SET fusion protein in cancer cells.

    PubMed

    Cho, Hyun-Soo; Kang, Jeong Gu; Lee, Jae-Hye; Lee, Jeong-Ju; Jeon, Seong Kook; Ko, Jeong-Heon; Kim, Dae-Soo; Park, Kun-Hyang; Kim, Yong-Sam; Kim, Nam-Soon

    2015-09-15

    TALE-nuclease chimeras (TALENs) can bind to and cleave specific genomic loci and, are used to engineer gene knockouts and additions. Recently, instead of using the FokI domain, epigenetically active domains, such as TET1 and LSD1, have been combined with TAL effector domains to regulate targeted gene expression via DNA and histone demethylation. However, studies of histone methylation in the TALE system have not been performed. Therefore, in this study, we established a novel targeted regulation system with a TAL effector domain and a histone methylation domain. To construct a TALE-methylation fusion protein, we combined a TAL effector domain containing an E-Box region to act as a Snail binding site and the SET domain of EHMT 2 to allow for histone methylation. The constructed TALE-SET module (TSET) repressed the expression of E-cadherin via by increasing H3K9 dimethylation. Moreover, the cells that overexpressed TSET showed increased cell migration and invasion. This is the first phenotype-based study of targeted histone methylation by the TALE module, and this new system can be applied in new cancer therapies to reduce side effects.

  9. Tales of future weather

    NASA Astrophysics Data System (ADS)

    Hazeleger, W.; van den Hurk, B. J. J. M.; Min, E.; van Oldenborgh, G. J.; Petersen, A. C.; Stainforth, D. A.; Vasileiadou, E.; Smith, L. A.

    2015-02-01

    Society is vulnerable to extreme weather events and, by extension, to human impacts on future events. As climate changes weather patterns will change. The search is on for more effective methodologies to aid decision-makers both in mitigation to avoid climate change and in adaptation to changes. The traditional approach uses ensembles of climate model simulations, statistical bias correction, downscaling to the spatial and temporal scales relevant to decision-makers, and then translation into quantities of interest. The veracity of this approach cannot be tested, and it faces in-principle challenges. Alternatively, numerical weather prediction models in a hypothetical climate setting can provide tailored narratives for high-resolution simulations of high-impact weather in a future climate. This 'tales of future weather' approach will aid in the interpretation of lower-resolution simulations. Arguably, it potentially provides complementary, more realistic and more physically consistent pictures of what future weather might look like.

  10. Two-axis angular effector

    DOEpatents

    Vaughn, Mark R.; Robinett, III, Rush D.; Phelan, John R.; Van Zuiden, Don M.

    1997-01-21

    A new class of coplanar two-axis angular effectors. These effectors combine a two-axis rotational joint analogous to a Cardan joint with linear actuators in a manner to produce a wider range of rotational motion about both axes defined by the joint. This new class of effectors also allows design of robotic manipulators having very high strength and efficiency. These effectors are particularly suited for remote operation in unknown surroundings, because of their extraordinary versatility. An immediate application is to the problems which arise in nuclear waste remediation.

  11. MorTAL Kombat: the story of defense against TAL effectors through loss-of-susceptibility

    PubMed Central

    Hutin, Mathilde; Pérez-Quintero, Alvaro L.; Lopez, Camilo; Szurek, Boris

    2015-01-01

    Many plant-pathogenic xanthomonads rely on Transcription Activator-Like (TAL) effectors to colonize their host. This particular family of type III effectors functions as specific plant transcription factors via a programmable DNA-binding domain. Upon binding to the promoters of plant disease susceptibility genes in a sequence-specific manner, the expression of these host genes is induced. However, plants have evolved specific strategies to counter the action of TAL effectors and confer resistance. One mechanism is to avoid the binding of TAL effectors by mutations of their DNA binding sites, resulting in resistance by loss-of-susceptibility. This article reviews our current knowledge of the susceptibility hubs targeted by Xanthomonas TAL effectors, possible evolutionary scenarios for plants to combat the pathogen with loss-of-function alleles, and how this knowledge can be used overall to develop new pathogen-informed breeding strategies and improve crop resistance. PMID:26236326

  12. Occupational health in fairy tales.

    PubMed

    Rivolta, Alice; Arienti, Federica; Smith, Derek R; Cesana, Giancarlo; Riva, Michele A

    2016-05-03

    Myths and folklore, as expressions of popular beliefs, provide valuable information on medical knowledge in earlier times. Fairy tales have often recounted occupational maladies throughout the ages and also provide some insight into the toxic effects of certain metals, such as mercury. Much historical information can be gleaned from unexpected sources, and as such, fairy tales should be more carefully scrutinized by contemporary researchers with an interest in the historical origins of workplace injury and disease.

  13. Fairy-tales in psychotherapy.

    PubMed

    Dieckmann, H

    1997-04-01

    Fairy-tales, like mythologies, can be found all over the world containing the same motif and chains of motifs. In this paper I have presented some theories on the occurrence of this archetypal phenomenon ranging from the old migration theory to Sheldrake's theory of morphogenetic fields. I have then tried to show how fairy-tale-motifs can appear in various ways in analytical therapy, often in hidden forms. We find them in patients' dreams as well as in their fantasies and associations. If the therapist is open to them they will also appear in his or her amplifications. He or she might then take note of the fairy-tale or point it out to the patient; in the latter case it might provide better access to the patient's problems and complexes as fairy-tales have an emotional completeness because of their pictorial character. Finally I have described the favourite fairy-tale of one of my patients and related it to his symptoms, his central complex and his personal ways of experiencing and behaving. This survey of how fairy-tales can be used in therapy with children and with adults far from exhaustive.

  14. Effector Mechanisms of Rejection

    PubMed Central

    Moreau, Aurélie; Varey, Emilie; Anegon, Ignacio; Cuturi, Maria-Cristina

    2013-01-01

    Organ transplantation appears today to be the best alternative to replace the loss of vital organs induced by various diseases. Transplants can, however, also be rejected by the recipient. In this review, we provide an overview of the mechanisms and the cells/molecules involved in acute and chronic rejections. T cells and B cells mainly control the antigen-specific rejection and act either as effector, regulatory, or memory cells. On the other hand, nonspecific cells such as endothelial cells, NK cells, macrophages, or polymorphonuclear cells are also crucial actors of transplant rejection. Last, beyond cells, the high contribution of antibodies, chemokines, and complement molecules in graft rejection is discussed in this article. The understanding of the different components involved in graft rejection is essential as some of them are used in the clinic as biomarkers to detect and quantify the level of rejection. PMID:24186491

  15. Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcription activator-like (TAL) effectors found in Xanthomonas spp. promote bacterial growth and plant susceptibility by binding specific DNA sequences or, effector-binding elements (EBEs), and inducing host gene expression. In this study, we have found substantially different transcriptional pro...

  16. SNARC Effect in Different Effectors.

    PubMed

    Hesse, Philipp N; Fiehler, Katja; Bremmer, Frank

    2016-01-01

    The SNARC (spatial numerical association of response codes) effect, indicating that subjects react faster to the left for small numbers and to the right for large numbers, is used as evidence for the idea that humans use space to organize number representations. Previous studies compared the SNARC effect across sensory modalities within participants and concluded modality independence. So far, it is unknown what sensory-to-motor mappings are involved in generating the SNARC effect and whether these mappings are identical for different effectors within subjects. Hence, we tested whether the SNARC effect is effector specific. Participants performed an auditory parity judgment task and responded with three different effectors: finger (button release), eyes (saccades), and arm (pointing). The SNARC effect occurred in each effector but varied in strength across the effectors. Across subjects, we found a significant correlation of SNARC strength for finger and arm responses suggesting the use of a shared sensory-to-motor mapping. SNARC strength did not correlate, however, between finger and eyes or arm and eyes. An additional statistical analysis based on conditional probabilities provided further evidence for SNARC-effector specificity. Taken together, our results suggest that the sensory-to-motor mapping is not as tight as it would be expected if the SNARC effect was effector independent.

  17. Potential Role of the Last Half Repeat in TAL Effectors Revealed by a Molecular Simulation Study

    PubMed Central

    Wan, Hua; Chang, Shan; Hu, Jian-ping; Tian, Xu-hong

    2016-01-01

    TAL effectors (TALEs) contain a modular DNA-binding domain that is composed of tandem repeats. In all naturally occurring TALEs, the end of tandem repeats is invariantly a truncated half repeat. To investigate the potential role of the last half repeat in TALEs, we performed comparative molecular dynamics simulations for the crystal structure of DNA-bound TALE AvrBs3 lacking the last half repeat and its modeled structure having the last half repeat. The structural stability analysis indicates that the modeled system is more stable than the nonmodeled system. Based on the principle component analysis, it is found that the AvrBs3 increases its structural compactness in the presence of the last half repeat. The comparison of DNA groove parameters of the two systems implies that the last half repeat also causes the change of DNA major groove binding efficiency. The following calculation of hydrogen bond reveals that, by stabilizing the phosphate binding with DNA at the C-terminus, the last half repeat helps to adopt a compact conformation at the protein-DNA interface. It further mediates more contacts between TAL repeats and DNA nucleotide bases. Finally, we suggest that the last half repeat is required for the high-efficient recognition of DNA by TALE. PMID:27803930

  18. Fairy Tales around the World. [Lesson Plan].

    ERIC Educational Resources Information Center

    2002

    Fairy tales are stories either created or strongly influenced by oral traditions. Their plots feature stark conflicts between good and evil, with magic and luck determining the usually happy endings. While each culture and geographic region of the world has its own body of folk tales and fairy tales, certain themes and motifs tend to be repeated…

  19. Ladrillo and Tales of Juan Bobo: Puerto Rican Folk Tales.

    ERIC Educational Resources Information Center

    Matos, Reinaldo; Matos, Ana

    These two illustrated elementary readers contain the Spanish and English versions of the Puerto Rican folk tales, "Ladrillo" and "Cuentos de Juan Bobo." They are part of a series of reading materials for elementary-level migrant children. These materials are intended to help the child relate to his culture, develop interest in…

  20. HANSEL AND GRETEL: A TALE OF TERROR.

    PubMed

    White, Robert S

    2015-10-01

    In the analysis of a woman with multiple childhood traumas, the fairy tale "Hansel and Gretel" figured prominently. The author discusses the use of the fairy tale in this case at various levels. He suggests an interplay between a national myth, the fairy tale, and a personal myth-the patient's psychodynamics. The fairy tale can be used to illuminate personal meanings derived from it. In the experience of childhood trauma, the repeated reading of a fairy tale can help organize and defend against terrifying anxiety.

  1. Genetic engineering of human pluripotent cells using TALE nucleases.

    PubMed

    Hockemeyer, Dirk; Wang, Haoyi; Kiani, Samira; Lai, Christine S; Gao, Qing; Cassady, John P; Cost, Gregory J; Zhang, Lei; Santiago, Yolanda; Miller, Jeffrey C; Zeitler, Bryan; Cherone, Jennifer M; Meng, Xiangdong; Hinkley, Sarah J; Rebar, Edward J; Gregory, Philip D; Urnov, Fyodor D; Jaenisch, Rudolf

    2011-07-07

    Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator-like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).

  2. Targeted mutagenesis in the malaria mosquito using TALE nucleases.

    PubMed

    Smidler, Andrea L; Terenzi, Olivier; Soichot, Julien; Levashina, Elena A; Marois, Eric

    2013-01-01

    Anopheles gambiae, the main mosquito vector of human malaria, is a challenging organism to manipulate genetically. As a consequence, reverse genetics studies in this disease vector have been largely limited to RNA interference experiments. Here, we report the targeted disruption of the immunity gene TEP1 using transgenic expression of Transcription-Activator Like Effector Nucleases (TALENs), and the isolation of several TEP1 mutant A. gambiae lines. These mutations inhibited protein production and rendered TEP1 mutants hypersusceptible to Plasmodium berghei. The TALEN technology opens up new avenues for genetic analysis in this disease vector and may offer novel biotechnology-based approaches for malaria control.

  3. Fairy Tales for Two Readers.

    ERIC Educational Resources Information Center

    Criscoe, Betty L., Ed.; Lanasa, Philip J., III, Ed.

    The 15 adapted fairy tales presented in this book were prepared for use in practicing oral reading by a parent and a child, a teacher and a child, or two children, one of whom reads slightly better than the other. The stories in the book are arranged in dialogue format for two readers. The high interest/low readability stories in the book are…

  4. "Arabian Tales": Standards of Learning.

    ERIC Educational Resources Information Center

    Brookshire, Cathy A.

    Virginia Standards of Learning for K-5 are listed in this paper with student activities related to observation of live theatre performances of "Arabian Tales" written and performed by the high school theater touring company, Organized Chaos. This play toured in Virginia in the academic year of 2000-2001. The play runs about 45 minutes.…

  5. A Tale of Four Electrons

    ERIC Educational Resources Information Center

    Burgmayer, Paul

    2011-01-01

    "A Tale of Four Electrons" is a creative writing assignment used with 10th-grade Honors Chemistry students. The project helps students consolidate their learning about bonding--an important unifying theme in chemistry--and answers questions such as (1) How are ionic, metallic, and covalent bonds related? (2) How do variations in electron…

  6. Number Crunching: A Sheep's Tale

    ERIC Educational Resources Information Center

    Sam, Chris Lam

    2005-01-01

    In this article, the author talks about an allegorical tale which he has written as a message for teachers of mathematics. The story is about Gordon, who led a flock of small sheep. Gordon was a mathematics genius; however, his flock criticized his teaching of numbers and his boring lectures. His furry-god-farmer advised him to share his…

  7. Folk Tales for Young Children.

    ERIC Educational Resources Information Center

    Ross, Ramon

    Most criticisms directed at basal reader stories concern the unreal characters which tend to depict only middle-class families and use unreal language patterns. However, such criticisms do not get to the core of the problem. A comparison of stories from basal readers with some famous folk tales illustrates how the here-and-now stories from basal…

  8. QueTAL: a suite of tools to classify and compare TAL effectors functionally and phylogenetically

    PubMed Central

    Pérez-Quintero, Alvaro L.; Lamy, Léo; Gordon, Jonathan L.; Escalon, Aline; Cunnac, Sébastien; Szurek, Boris; Gagnevin, Lionel

    2015-01-01

    Transcription Activator-Like (TAL) effectors from Xanthomonas plant pathogenic bacteria can bind to the promoter region of plant genes and induce their expression. DNA-binding specificity is governed by a central domain made of nearly identical repeats, each determining the recognition of one base pair via two amino acid residues (a.k.a. Repeat Variable Di-residue, or RVD). Knowing how TAL effectors differ from each other within and between strains would be useful to infer functional and evolutionary relationships, but their repetitive nature precludes reliable use of traditional alignment methods. The suite QueTAL was therefore developed to offer tailored tools for comparison of TAL effector genes. The program DisTAL considers each repeat as a unit, transforms a TAL effector sequence into a sequence of coded repeats and makes pair-wise alignments between these coded sequences to construct trees. The program FuncTAL is aimed at finding TAL effectors with similar DNA-binding capabilities. It calculates correlations between position weight matrices of potential target DNA sequence predicted from the RVD sequence, and builds trees based on these correlations. The programs accurately represented phylogenetic and functional relationships between TAL effectors using either simulated or literature-curated data. When using the programs on a large set of TAL effector sequences, the DisTAL tree largely reflected the expected species phylogeny. In contrast, FuncTAL showed that TAL effectors with similar binding capabilities can be found between phylogenetically distant taxa. This suite will help users to rapidly analyse any TAL effector genes of interest and compare them to other available TAL genes and should improve our understanding of TAL effectors evolution. It is available at http://bioinfo-web.mpl.ird.fr/cgi-bin2/quetal/quetal.cgi. PMID:26284082

  9. Oomycetes, effectors, and all that jazz.

    PubMed

    Bozkurt, Tolga O; Schornack, Sebastian; Banfield, Mark J; Kamoun, Sophien

    2012-08-01

    Plant pathogenic oomycetes secrete a diverse repertoire of effector proteins that modulate host innate immunity and enable parasitic infection. Understanding how effectors evolve, translocate and traffic inside host cells, and perturb host processes are major themes in the study of oomycete-plant interactions. The last year has seen important progress in the study of oomycete effectors with, notably, the elucidation of the 3D structures of five RXLR effectors, and novel insights into how cytoplasmic effectors subvert host cells. In this review, we discuss these and other recent advances and highlight the most important open questions in oomycete effector biology.

  10. Effector proteins of rust fungi

    PubMed Central

    Petre, Benjamin; Joly, David L.; Duplessis, Sébastien

    2014-01-01

    Rust fungi include many species that are devastating crop pathogens. To develop resistant plants, a better understanding of rust virulence factors, or effector proteins, is needed. Thus far, only six rust effector proteins have been described: AvrP123, AvrP4, AvrL567, AvrM, RTP1, and PGTAUSPE-10-1. Although some are well established model proteins used to investigate mechanisms of immune receptor activation (avirulence activities) or entry into plant cells, how they work inside host tissues to promote fungal growth remains unknown. The genome sequences of four rust fungi (two Melampsoraceae and two Pucciniaceae) have been analyzed so far. Genome-wide analyses of these species, as well as transcriptomics performed on a broader range of rust fungi, revealed hundreds of small secreted proteins considered as rust candidate secreted effector proteins (CSEPs). The rust community now needs high-throughput approaches (effectoromics) to accelerate effector discovery/characterization and to better understand how they function in planta. However, this task is challenging due to the non-amenability of rust pathosystems (obligate biotrophs infecting crop plants) to traditional molecular genetic approaches mainly due to difficulties in culturing these species in vitro. The use of heterologous approaches should be promoted in the future. PMID:25191335

  11. Improving a Gripper End Effector

    SciTech Connect

    Mullen, O Dennis; Smith, Christopher M.; Gervais, Kevin L.

    2001-01-31

    This paper discusses the improvement made to an existing four-bar linkage gripping end effector to adapt it for use in a current project. The actuating linkage was modified to yield higher jaw force overall and particularly in the critical range of jaw displacement

  12. Folk Tales and Fairy Tales: Literature Curriculum C-D [Grades Three and Four]; Teacher's Guide.

    ERIC Educational Resources Information Center

    Oregon Univ., Eugene. Oregon Elementary English Project.

    This curriculum guide is intended to introduce elementary school students to folktales and fairy tales. Three categories of tales, each containing four examples, are included: "Encounters with Wee Folk,""Foolish Use of Wishes," and "Unlikely Success." The folktales and fairy tales in this unit are described as…

  13. Dynamic Aspects of Fairy Tales: Social and Emotional Competence through Fairy Tales.

    ERIC Educational Resources Information Center

    Hohr, Hansjorg

    2000-01-01

    Explores how fairy tales address socio-emotional challenges children face during their socialization. Applies a structural theory of fairy tales to three literary versions of the Cinderella story. Suggests that the combination of simplicity of form and complexity of content makes the fairy tale a powerful tool for perception of and reflection on…

  14. The type III effectors of Xanthomonas.

    PubMed

    White, Frank F; Potnis, Neha; Jones, Jeffrey B; Koebnik, Ralf

    2009-11-01

    A review of type III effectors (T3 effectors) from strains of Xanthomonas reveals a growing list of candidate and known effectors based on functional assays and sequence and structural similarity searches of genomic data. We propose that the effectors and suspected effectors should be distributed into 39 so-called Xop groups reflecting sequence similarity. Some groups have structural motifs for putative enzymatic functions, and recent studies have provided considerable insight into the interaction with host factors in their function as mediators of virulence and elicitors of resistance for a few specific T3 effectors. Many groups are related to T3 effectors of plant and animal pathogenic bacteria, and several groups appear to have been exploited primarily by Xanthomonas species based on available data. At the same time, a relatively large number of candidate effectors remain to be examined in more detail with regard to their function within host cells.

  15. EffectorP: predicting fungal effector proteins from secretomes using machine learning.

    PubMed

    Sperschneider, Jana; Gardiner, Donald M; Dodds, Peter N; Tini, Francesco; Covarelli, Lorenzo; Singh, Karam B; Manners, John M; Taylor, Jennifer M

    2016-04-01

    Eukaryotic filamentous plant pathogens secrete effector proteins that modulate the host cell to facilitate infection. Computational effector candidate identification and subsequent functional characterization delivers valuable insights into plant-pathogen interactions. However, effector prediction in fungi has been challenging due to a lack of unifying sequence features such as conserved N-terminal sequence motifs. Fungal effectors are commonly predicted from secretomes based on criteria such as small size and cysteine-rich, which suffers from poor accuracy. We present EffectorP which pioneers the application of machine learning to fungal effector prediction. EffectorP improves fungal effector prediction from secretomes based on a robust signal of sequence-derived properties, achieving sensitivity and specificity of over 80%. Features that discriminate fungal effectors from secreted noneffectors are predominantly sequence length, molecular weight and protein net charge, as well as cysteine, serine and tryptophan content. We demonstrate that EffectorP is powerful when combined with in planta expression data for predicting high-priority effector candidates. EffectorP is the first prediction program for fungal effectors based on machine learning. Our findings will facilitate functional fungal effector studies and improve our understanding of effectors in plant-pathogen interactions. EffectorP is available at http://effectorp.csiro.au.

  16. The Epistemic Value of Cautionary Tales

    ERIC Educational Resources Information Center

    Shields, William M.

    2006-01-01

    A cautionary tale has become something of a cottage industry in the past decade. To be sure, there has been plenty of material for these publications: Bhopal, Chernobyl, "Exxon Valdez," Three Mile Island, and "Challenger" have entered the lexicon as virtual synonyms for "disaster." Cautionary tale scenarios involve a different kind of causality.…

  17. "Turtle Island Tales." Cue Sheet for Students.

    ERIC Educational Resources Information Center

    Carr, Gail

    This performance guide is designed for teachers to use with students before and after a shadow play performance of "Turtle Island Tales" by Hobey Ford and His Golden Rod Puppets. The guide, called a "Cuesheet," contains seven activity sheets for use in class, addressing: (1) The Tales (offering brief outlines of the three tales…

  18. Children's and Adults' Responses to Fairy Tales.

    ERIC Educational Resources Information Center

    Strayer, Janet

    1995-01-01

    Investigated North American children's and adults' familiarity with, and liking for, fairy tales. Results support the hypotheses that children's liking for fairy tales relates significantly to their involvement in imagining activities and that more general imagining involvements are likely to be associated with, or to mediate differences in, the…

  19. Pourquoi Tales on the Literacy Stage

    ERIC Educational Resources Information Center

    Foster, Karen K.; Theiss, Deb; Buchanan-Butterfield, Dawna Lisa

    2008-01-01

    Since ancient times, humans have sought explanations for the mysteries of nature's beauty and variety. Like other kinds of folk stories, pourquoi tales can be considered parables about how humans and animals originated or why they look or behave the way they do. Pourquoi tales have high appeal, are generally simple and straightforward in…

  20. Happily Ever After? Teens and Fairy Tales.

    ERIC Educational Resources Information Center

    Tuccillo, Diane P.

    2001-01-01

    Argues, from the author's experience and with supporting quotes from many teenagers, that modern retellings of classic fairy tales can be very popular with teenage readers. Discusses numerous such stories, and offers an extensive list of retold fairy tales, organized into: Cinderella stories, Beauty and the Beast stories, Sleeping Beauty stories,…

  1. A surrealistic tale of electricity

    NASA Astrophysics Data System (ADS)

    Fuchs, Hans U.

    1986-10-01

    Using thermodynamics as an analogy, an ``electricity machine'' is constructed and analyzed. This machine runs through a ``Carnot'' cycle. The analysis leads to the construction of a ``new'' quantity called ``reduced electricity'' (charge). The example shows what life in electrodynamics would be without the substancelike quantity which we call electrical charge. (This state of affairs is accepted in thermodynamics.) Imagine the development of the theory of electricity had fallen into the hands of the thermodynamicists. As fairy and other tales go, they have an air of surrealism surrounding them.

  2. TAL effectors and activation of predicted host targets distinguish Asian from African strains of the rice pathogen Xanthomonas oryzae pv. oryzicola while strict conservation suggests universal importance of five TAL effectors.

    PubMed

    Wilkins, Katherine E; Booher, Nicholas J; Wang, Li; Bogdanove, Adam J

    2015-01-01

    Xanthomonas oryzae pv. oryzicola (Xoc) causes the increasingly important disease bacterial leaf streak of rice (BLS) in part by type III delivery of repeat-rich transcription activator-like (TAL) effectors to upregulate host susceptibility genes. By pathogen whole genome, single molecule, real-time sequencing and host RNA sequencing, we compared TAL effector content and rice transcriptional responses across 10 geographically diverse Xoc strains. TAL effector content is surprisingly conserved overall, yet distinguishes Asian from African isolates. Five TAL effectors are conserved across all strains. In a prior laboratory assay in rice cv. Nipponbare, only two contributed to virulence in strain BLS256 but the strict conservation indicates all five may be important, in different rice genotypes or in the field. Concatenated and aligned, TAL effector content across strains largely reflects relationships based on housekeeping genes, suggesting predominantly vertical transmission. Rice transcriptional responses did not reflect these relationships, and on average, only 28% of genes upregulated and 22% of genes downregulated by a strain are up- and down- regulated (respectively) by all strains. However, when only known TAL effector targets were considered, the relationships resembled those of the TAL effectors. Toward identifying new targets, we used the TAL effector-DNA recognition code to predict effector binding elements in promoters of genes upregulated by each strain, but found that for every strain, all upregulated genes had at least one. Filtering with a classifier we developed previously decreases the number of predicted binding elements across the genome, suggesting that it may reduce false positives among upregulated genes. Applying this filter and eliminating genes for which upregulation did not strictly correlate with presence of the corresponding TAL effector, we generated testable numbers of candidate targets for four of the five strictly conserved TAL

  3. TAL effectors and activation of predicted host targets distinguish Asian from African strains of the rice pathogen Xanthomonas oryzae pv. oryzicola while strict conservation suggests universal importance of five TAL effectors

    PubMed Central

    Wilkins, Katherine E.; Booher, Nicholas J.; Wang, Li; Bogdanove, Adam J.

    2015-01-01

    Xanthomonas oryzae pv. oryzicola (Xoc) causes the increasingly important disease bacterial leaf streak of rice (BLS) in part by type III delivery of repeat-rich transcription activator-like (TAL) effectors to upregulate host susceptibility genes. By pathogen whole genome, single molecule, real-time sequencing and host RNA sequencing, we compared TAL effector content and rice transcriptional responses across 10 geographically diverse Xoc strains. TAL effector content is surprisingly conserved overall, yet distinguishes Asian from African isolates. Five TAL effectors are conserved across all strains. In a prior laboratory assay in rice cv. Nipponbare, only two contributed to virulence in strain BLS256 but the strict conservation indicates all five may be important, in different rice genotypes or in the field. Concatenated and aligned, TAL effector content across strains largely reflects relationships based on housekeeping genes, suggesting predominantly vertical transmission. Rice transcriptional responses did not reflect these relationships, and on average, only 28% of genes upregulated and 22% of genes downregulated by a strain are up- and down- regulated (respectively) by all strains. However, when only known TAL effector targets were considered, the relationships resembled those of the TAL effectors. Toward identifying new targets, we used the TAL effector-DNA recognition code to predict effector binding elements in promoters of genes upregulated by each strain, but found that for every strain, all upregulated genes had at least one. Filtering with a classifier we developed previously decreases the number of predicted binding elements across the genome, suggesting that it may reduce false positives among upregulated genes. Applying this filter and eliminating genes for which upregulation did not strictly correlate with presence of the corresponding TAL effector, we generated testable numbers of candidate targets for four of the five strictly conserved TAL

  4. Familiar Fairy Tale Picture Books Transformed into Teen Novels.

    ERIC Educational Resources Information Center

    Chance, Rosemary

    2003-01-01

    Describes characteristics of fairy tales. Discusses use of fairy tales and novels for teens in the classroom. Presents annotations of 31 titles including both picture books and young adult novels grouped by nine popular tales. Concludes that through comparing picture books and teen novels, there is one last chance to introduce fairy tales to older…

  5. Marker for type VI secretion system effectors

    PubMed Central

    Salomon, Dor; Kinch, Lisa N.; Trudgian, David C.; Guo, Xiaofeng; Klimko, John A.; Grishin, Nick V.; Mirzaei, Hamid; Orth, Kim

    2014-01-01

    Bacteria use diverse mechanisms to kill, manipulate, and compete with other cells. The recently discovered type VI secretion system (T6SS) is widespread in bacterial pathogens and used to deliver virulence effector proteins into target cells. Using comparative proteomics, we identified two previously unidentified T6SS effectors that contained a conserved motif. Bioinformatic analyses revealed that this N-terminal motif, named MIX (marker for type six effectors), is found in numerous polymorphic bacterial proteins that are primarily located in the T6SS genome neighborhood. We demonstrate that several MIX-containing proteins are T6SS effectors and that they are not required for T6SS activity. Thus, we propose that MIX-containing proteins are T6SS effectors. Our findings allow for the identification of numerous uncharacterized T6SS effectors that will undoubtedly lead to the discovery of new biological mechanisms. PMID:24927539

  6. Space Station end effector strategy study

    NASA Technical Reports Server (NTRS)

    Katzberg, Stephen J.; Jensen, Robert L.; Willshire, Kelli F.; Satterthwaite, Robert E.

    1987-01-01

    The results of a study are presented for terminology definition, identification of functional requirements, technolgy assessment, and proposed end effector development strategies for the Space Station Program. The study is composed of a survey of available or under-developed end effector technology, identification of requirements from baselined Space Station documents, a comparative assessment of the match between technology and requirements, and recommended strategies for end effector development for the Space Station Program.

  7. Anthropology: The Long Lives of Fairy Tales.

    PubMed

    Pagel, Mark

    2016-04-04

    Anthropologists, borrowing techniques from evolutionary biology, have demonstrated that some common fairy tales can be traced back 5,000 years, or more, long before the development of written traditions.

  8. Orbital maneuvering vehicle end effectors

    NASA Technical Reports Server (NTRS)

    Myers, W. Neill (Inventor); Forbes, John C. (Inventor); Barnes, Wayne L. (Inventor)

    1988-01-01

    An end effector device (A) for grasping and holding an article such as a handle (18) of a space telescope is disclosed. The device includes a V-shaped capture window (74) defined as inclined surfaces (76, 78) in parallel face plates (22, 24) which converge toward a retainer recess (54) in which the handle is retained. A pivotal finger (30) meshes with a pair of pivoted fingers (26, 28) which rotate in counterrotation. The fingers rotate to pull a handle within the capture window into recess (54) where latches (50) lock handle (18) in the recess. To align the capture window, plates (22, 24) may be cocked plus or minus five degrees on base (64).

  9. A tale of two Paks.

    PubMed

    Arias-Romero, Luis E; Chernoff, Jonathan

    2008-02-01

    Paks (p21-activated kinases) are effectors for the small GTPases Cdc42 (cell division cycle 42) and Rac that play fundamental roles in a wide range of cellular processes, including cell morphology, motility, survival, gene transcription, apoptosis and hormone signalling. These enzymes are widely expressed in numerous tissues and are activated by extracellular signals through GTPase-dependent and -independent mechanisms. On the basis of structural and biochemical features, the Pak family members have been classified into two groups, comprising three members each. The two Pak groups have generally been considered as two halves of a single entity, but accumulating evidence indicates that this is not so. In this review, we discuss recent advances in our understanding of the structure, regulation and function of these kinases, emphasizing the many differences between these two groups of signalling proteins.

  10. A Fairy-Tale Landscape

    NASA Technical Reports Server (NTRS)

    2008-01-01

    [figure removed for brevity, see original site] Click on image for animation

    Fun, fairy-tale nicknames have been assigned to features in this animated view of the workspace reachable by the robotic arm of NASA's Phoenix Mars Lander. For example, 'Sleepy Hollow' denotes a trench and 'Headless' designates a rock.

    A 'National Park,' marked by purple text and a purple arrow, has been set aside for protection until scientists and engineers have tested the operation of the robotic scoop. First touches with the scoop will be to the left of the 'National Park' line.

    Scientists use such informal names for easy identification of features of interest during the mission.

    In this view, rocks are circled in yellow, other areas of interest in green. The images were taken by the lander's 7-foot mast camera, called the Surface Stereo Imager.

    The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  11. ROBOTIC TANK INSPECTION END EFFECTOR

    SciTech Connect

    Rachel Landry

    1999-10-01

    The objective of this contract between Oceaneering Space Systems (OSS) and the Department of Energy (DOE) was to provide a tool for the DOE to inspect the inside tank walls of underground radioactive waste storage tanks in their tank farms. Some of these tanks are suspected to have leaks, but the harsh nature of the environment within the tanks precludes human inspection of tank walls. As a result of these conditions only a few inspection methods can fulfill this task. Of the methods available, OSS chose to pursue Alternating Current Field Measurement (ACFM), because it does not require clean surfaces for inspection, nor any contact with the Surface being inspected, and introduces no extra by-products in the inspection process (no coupling fluids or residues are left behind). The tool produced by OSS is the Robotic Tank Inspection End Effector (RTIEE), which is initially deployed on the tip of the Light Duty Utility Arm (LDUA). The RTEE combines ACFM with a color video camera for both electromagnetic and visual inspection The complete package consists of an end effector, its corresponding electronics and software, and a user's manual to guide the operator through an inspection. The system has both coarse and fine inspection modes and allows the user to catalog defects and suspected areas of leakage in a database for further examination, which may lead to emptying the tank for repair, decommissioning, etc.. The following is an updated report to OSS document OSS-21100-7002, which was submitted in 1995. During the course of the contract, two related subtasks arose, the Wall and Coating Thickness Sensor and the Vacuum Scarifying and Sampling Tool Assembly. The first of these subtasks was intended to evaluate the corrosion and wall thinning of 55-gallon steel drums. The second was retrieved and characterized the waste material trapped inside the annulus region of the underground tanks on the DOE's tank farms. While these subtasks were derived from the original intent of

  12. Oxysterols and Their Cellular Effectors

    PubMed Central

    Olkkonen, Vesa M.; Béaslas, Olivier; Nissilä, Eija

    2012-01-01

    Oxysterols are oxidized 27-carbon cholesterol derivatives or by-products of cholesterol biosynthesis, with a spectrum of biologic activities. Several oxysterols have cytotoxic and pro-apoptotic activities, the ability to interfere with the lateral domain organization, and packing of membrane lipids. These properties may account for their suggested roles in the pathology of diseases such as atherosclerosis, age-onset macular degeneration and Alzheimer’s disease. Oxysterols also have the capacity to induce inflammatory responses and play roles in cell differentiation processes. The functions of oxysterols as intermediates in the synthesis of bile acids and steroid hormones, and as readily transportable forms of sterol, are well established. Furthermore, their actions as endogenous regulators of gene expression in lipid metabolism via liver X receptors and the Insig (insulin-induced gene) proteins have been investigated in detail. The cytoplasmic oxysterol-binding protein (OSBP) homologues form a group of oxysterol/cholesterol sensors that has recently attracted a lot of attention. However, their mode of action is, as yet, poorly understood. Retinoic acid receptor-related orphan receptors (ROR) α and γ, and Epstein-Barr virus induced gene 2 (EBI2) have been identified as novel oxysterol receptors, revealing new physiologic oxysterol effector mechanisms in development, metabolism, and immunity, and evoking enhanced interest in these compounds in the field of biomedicine. PMID:24970128

  13. Five phylogenetically close rice SWEET genes confer TAL effector-mediated susceptibility to Xanthomonas oryzae pv. oryzae.

    PubMed

    Streubel, Jana; Pesce, Céline; Hutin, Mathilde; Koebnik, Ralf; Boch, Jens; Szurek, Boris

    2013-11-01

    Bacterial plant-pathogenic Xanthomonas strains translocate transcription activator-like (TAL) effectors into plant cells to function as specific transcription factors. Only a few plant target genes of TAL effectors have been identified, so far. Three plant SWEET genes encoding putative sugar transporters are known to be induced by TAL effectors from rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo). We predict and validate that expression of OsSWEET14 is induced by a novel TAL effector, Tal5, from an African Xoo strain. Artificial TAL effectors (ArtTALs) were constructed to individually target 20 SWEET orthologs in rice. They were used as designer virulence factors to study which rice SWEET genes can support Xoo virulence. The Tal5 target box differs from those of the already known TAL effectors TalC, AvrXa7 and PthXo3, which also induce expression of OsSWEET14, suggesting evolutionary convergence on key targets. ArtTALs efficiently complemented an Xoo talC mutant, demonstrating that specific induction of OsSWEET14 is the key target of TalC. ArtTALs that specifically target individual members of the rice SWEET family revealed three known and two novel SWEET genes to support bacterial virulence. Our results demonstrate that five phylogenetically close SWEET proteins, which presumably act as sucrose transporters, can support Xoo virulence.

  14. Epigenetic control of effectors in plant pathogens.

    PubMed

    Gijzen, Mark; Ishmael, Chelsea; Shrestha, Sirjana D

    2014-01-01

    Plant pathogens display impressive versatility in adapting to host immune systems. Pathogen effector proteins facilitate disease but can become avirulence (Avr) factors when the host acquires discrete recognition capabilities that trigger immunity. The mechanisms that lead to changes to pathogen Avr factors that enable escape from host immunity are diverse, and include epigenetic switches that allow for reuse or recycling of effectors. This perspective outlines possibilities of how epigenetic control of Avr effector gene expression may have arisen and persisted in filamentous plant pathogens, and how it presents special problems for diagnosis and detection of specific pathogen strains or pathotypes.

  15. Mycobacterium tuberculosis effectors interfering host apoptosis signaling.

    PubMed

    Liu, Minqiang; Li, Wu; Xiang, Xiaohong; Xie, Jianping

    2015-07-01

    Tuberculosis remains a serious human public health concern. The coevolution between its pathogen Mycobacterium tuberculosis and human host complicated the way to prevent and cure TB. Apoptosis plays subtle role in this interaction. The pathogen endeavors to manipulate the apoptosis via diverse effectors targeting key signaling nodes. In this paper, we summarized the effectors pathogen used to subvert the apoptosis, such as LpqH, ESAT-6/CFP-10, LAMs. The interplay between different forms of cell deaths, such as apoptosis, autophagy, necrosis, is also discussed with a focus on the modes of action of effectors, and implications for better TB control.

  16. Introducing Children to Folk Tales. Bill Harp Professional Teachers Library.

    ERIC Educational Resources Information Center

    Weir, Beth

    This book provides K-8 teachers with an introductory resource on folk tales. The book acknowledges that teachers are often very interested in folk tales but lack the time to research them. Each chapter contains some background information on a story type or a character. The book's six chapters are as follows: (1) "The Folk Tale Tradition"; (2)…

  17. Using Fairy Tales To Generate High Interest in Short Fiction.

    ERIC Educational Resources Information Center

    McKenna, Barbara J.

    2001-01-01

    Discusses how fairy tales provide the vehicle by which students become genuinely engaged in reading and writing short fiction. Outlines a three-step process moving students from familiar stories, to variations on traditional tales, finally to new stories. Details how writing, revising, illustrating, and binding a fairy tale engages students. (PM)

  18. Through Fairy-Tales to Math in the Lessons

    ERIC Educational Resources Information Center

    Andersone, Rudite

    2009-01-01

    Every person in his lifetime has many times experienced the miraculous impact of the fairy-tale on his imagination, mind and feelings. But we seldom reflect upon the content not only in the ethical aspect of fairy-tales but also as a source of different kinds of knowledge, among them the knowledge of mathematics. The use of fairy-tales in lessons…

  19. Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases.

    PubMed

    Watanabe, Takahito; Ochiai, Hiroshi; Sakuma, Tetsushi; Horch, Hadley W; Hamaguchi, Naoya; Nakamura, Taro; Bando, Tetsuya; Ohuchi, Hideyo; Yamamoto, Takashi; Noji, Sumihare; Mito, Taro

    2012-01-01

    Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.

  20. Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases

    PubMed Central

    Watanabe, Takahito; Ochiai, Hiroshi; Sakuma, Tetsushi; Horch, Hadley W.; Hamaguchi, Naoya; Nakamura, Taro; Bando, Tetsuya; Ohuchi, Hideyo; Yamamoto, Takashi; Noji, Sumihare; Mito, Taro

    2012-01-01

    Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy. PMID:22910363

  1. Jet Engine Exhaust Nozzle Flow Effector

    NASA Technical Reports Server (NTRS)

    Turner, Travis L. (Inventor); Cano, Roberto J. (Inventor); Silox, Richard J. (Inventor); Buehrle, Ralph D. (Inventor); Cagle, Christopher M. (Inventor); Cabell, Randolph H. (Inventor); Hilton, George C. (Inventor)

    2014-01-01

    A jet engine exhaust nozzle flow effector is a chevron formed with a radius of curvature with surfaces of the flow effector being defined and opposing one another. At least one shape memory alloy (SMA) member is embedded in the chevron closer to one of the chevron's opposing surfaces and substantially spanning from at least a portion of the chevron's root to the chevron's tip.

  2. Jet Engine Exhaust Nozzle Flow Effector

    NASA Technical Reports Server (NTRS)

    Turner, Travis L. (Inventor); Cano, Roberto J. (Inventor); Silcox, Richard J. (Inventor); Buehrle, Ralph D. (Inventor); Cagle, Christopher M. (Inventor); Cabell, Randolph H. (Inventor); Hilton, George C. (Inventor)

    2011-01-01

    A jet engine exhaust nozzle flow effector is a chevron formed with a radius of curvature with surfaces of the flow effector being defined and opposing one another. At least one shape memory alloy (SMA) member is embedded in the chevron closer to one of the chevron's opposing surfaces and substantially spanning from at least a portion of the chevron's root to the chevron's tip.

  3. Fairy tales as a trance experience: possible therapeutic uses.

    PubMed

    Stevens-Guille, M E; Boersma, F J

    1992-04-01

    The psychological literature contains little documentation of the therapeutic use of fairy tales. We suggest that fairy tales are uniquely suitable for hypnotherapy and for helping clients reframe existential issues. We propose that the structure of fairy tales allows the meaning of the story to be applied personally and that they also stimulate unconscious search. We examine the way in which hypnosis is achieved when fairy tales are read to children, as well as possible therapeutic uses of this learning set in therapy with both children and adults. We conclude by suggesting that fairy tales need to be given serious consideration as an alternative therapeutic trance procedure.

  4. Fairy Tale Retellings between Art and Pedagogy

    ERIC Educational Resources Information Center

    Joosen, Vanessa

    2005-01-01

    In this article, it is shown how authors of fairy tale retellings have incorporated ideas of feminist literary criticism into a fictional form. As such, these retellings display the tension between the pedagogic and aesthetic aspects of all children's literature. Jane Yolen's "Sleeping Ugly" is chosen as a case study: although it can be argued…

  5. Speech, Image, Action: Animating Tales from Shakespeare.

    ERIC Educational Resources Information Center

    Bottoms, Janet

    2001-01-01

    Challenges two assumptions: that children are naturally disposed toward the animated cartoon, and that translating Shakespeare's plays into this medium automatically simplifies and gives them child appeal. Examines the confusions and cross-purposes that surrounded the making of the "Animated Tales" videos, and argues that there are…

  6. Exploring World Cultures through Folk Tales.

    ERIC Educational Resources Information Center

    Dennis, Sarah

    Providing students with the opportunity to study different cultures increases their global understanding and helps foster tolerance of cultural differences. In this lesson, grade 3-5 students read folk tales from Japan, Wales, and Africa and depict the stories visually for purposes of retelling. Students also research the countries and share a…

  7. 1972 Witches' Tales Around Reading Problems.

    ERIC Educational Resources Information Center

    Jampolsky, Gerald G.

    This paper describes how some children with learning problems feel about themselves; points out some possible witches' tales regarding understanding and communication with such children; and discusses some clinical opinions that have been developed at the Child Center, Kentfield, regarding these children. The contents of the paper include: a…

  8. "Clockwork": Philip Pullman's Posthuman Fairy Tale

    ERIC Educational Resources Information Center

    Gooding, Richard

    2011-01-01

    This article examines the connections between posthumanism and narrative form in Philip Pullman's "Clockwork." Beginning with an account of Pullman's materialism, it argues that the novel represents consciousness and agency as emergent properties of matter, a position that manifests itself first in the tale's figurative language and later in the…

  9. Bacterial Effector Nanoparticles as Breast Cancer Therapeutics.

    PubMed

    Herrera Estrada, Lina; Padmore, Trudy J; Champion, Julie A

    2016-03-07

    Bacterial pathogens trigger cell death by a variety of mechanisms, including injection of effector proteins. Effector proteins have great potential as anticancer agents because they efficiently subvert a variety of eukaryotic signaling pathways involved in cancer development, drug resistance, and metastasis. In breast cancer, MAPK and NFκB pathways are known to be dysregulated. YopJ, an effector from Yersinia pestis, downregulates MAPK and NFκB pathways to induce cell death in specific cell types. We expressed YopJ in Escherichia coli as a fusion protein with glutathione S-transferase (GST), forming self-assembled protein nanoparticles with diameters of 100 nm. YopJ-GST nanoparticles efficiently delivered protein to cells, replacing the need for the pathogen secretion mechanism for effector delivery to cells. These nanoparticles induced dose and time dependent death in SKBR-3 breast cancer cells. After 72 h, 97% of cells died, significantly more than with the same molar dose of doxorubicin. Treatment with sublethal doses of nanoparticles decreased cell migration in vitro and downregulated the MAPK ERK 1/2 pathway, which has been correlated to metastasis. Exposure to a panel of breast cancer cell lines showed that YopJ-GST nanoparticles are cytotoxic to different subtypes, including doxorubicin resistant cells. However, they were not cytotoxic to NIH/3T3 fibroblasts or HeLa cells. Thus, YopJ-GST nanoparticles demonstrate the potential of effector proteins as breast cancer therapeutics with selective cytotoxicity and the capacity to decrease metastatic predictive behaviors.

  10. End effector with astronaut foot restraint

    NASA Technical Reports Server (NTRS)

    Monford, Leo G., Jr. (Inventor)

    1991-01-01

    The combination of a foot restraint platform designed primarily for use by an astronaut being rigidly and permanently attached to an end effector which is suitable for attachment to the manipulator arm of a remote manipulating system is described. The foot restraint platform is attached by a brace to the end effector at a location away from the grappling interface of the end effector. The platform comprises a support plate provided with a pair of stirrups for receiving the toe portion of an astronaut's boots when standing on the platform and a pair of heel retainers in the form of raised members which are fixed to the surface of the platform and located to provide abutment surfaces for abutting engagement with the heels of the astronaut's boots when his toes are in the stirrups. The heel retainers preclude a backward sliding movement of the feet on the platform and instead require a lifting of the heels in order to extract the feet. The brace for attaching the foot restraint platform to the end effector may include a pivot or swivel joint to permit various orientations of the platform with respect to the end effector.

  11. A library of TAL effector nucleases spanning the human genome.

    PubMed

    Kim, Yongsub; Kweon, Jiyeon; Kim, Annie; Chon, Jae Kyung; Yoo, Ji Yeon; Kim, Hye Joo; Kim, Sojung; Lee, Choongil; Jeong, Euihwan; Chung, Eugene; Kim, Doyoung; Lee, Mi Seon; Go, Eun Mi; Song, Hye Jung; Kim, Hwangbeom; Cho, Namjin; Bang, Duhee; Kim, Seokjoong; Kim, Jin-Soo

    2013-03-01

    Transcription activator-like (TAL) effector nucleases (TALENs) can be readily engineered to bind specific genomic loci, enabling the introduction of precise genetic modifications such as gene knockouts and additions. Here we present a genome-scale collection of TALENs for efficient and scalable gene targeting in human cells. We chose target sites that did not have highly similar sequences elsewhere in the genome to avoid off-target mutations and assembled TALEN plasmids for 18,740 protein-coding genes using a high-throughput Golden-Gate cloning system. A pilot test involving 124 genes showed that all TALENs were active and disrupted their target genes at high frequencies, although two of these TALENs became active only after their target sites were partially demethylated using an inhibitor of DNA methyltransferase. We used our TALEN library to generate single- and double-gene-knockout cells in which NF-κB signaling pathways were disrupted. Compared with cells treated with short interfering RNAs, these cells showed unambiguous suppression of signal transduction.

  12. Waking up dormant tumor suppressor genes with zinc fingers, TALEs and the CRISPR/dCas9 system

    PubMed Central

    Garcia-Bloj, Benjamin; Moses, Colette; Sgro, Agustin; Plani-Lam, Janice; Arooj, Mahira; Duffy, Ciara; Thiruvengadam, Shreyas; Sorolla, Anabel; Rashwan, Rabab; Mancera, Ricardo L.; Leisewitz, Andrea; Swift-Scanlan, Theresa; Corvalan, Alejandro H.; Blancafort, Pilar

    2016-01-01

    The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases. PMID:27528034

  13. RXLR effectors of plant pathogenic oomycetes.

    PubMed

    Morgan, William; Kamoun, Sophien

    2007-08-01

    Oomycetes are a phylogenetically distinct group of organisms that include some of the most devastating plant pathogens. Recent characterization of four oomycete Avr genes revealed that they encode effector proteins with a common modular structure, including a N-terminal conserved RXLR motif. Several lines of evidence initially indicated, with support from more recent works, that these Avr proteins are secreted by the pathogen and then translocated into the host cell during infection. In addition to elucidating the machinery required for host-cell transport, future works remain to determine the myriad virulence functions of oomycete RXLR effector proteins.

  14. SAPTA: a new design tool for improving TALE nuclease activity.

    PubMed

    Lin, Yanni; Fine, Eli J; Zheng, Zhilan; Antico, Christopher J; Voit, Richard A; Porteus, Matthew H; Cradick, Thomas J; Bao, Gang

    2014-04-01

    Transcription activator-like effector nucleases (TALENs) have become a powerful tool for genome editing due to the simple code linking the amino acid sequences of their DNA-binding domains to TALEN nucleotide targets. While the initial TALEN-design guidelines are very useful, user-friendly tools defining optimal TALEN designs for robust genome editing need to be developed. Here we evaluated existing guidelines and developed new design guidelines for TALENs based on 205 TALENs tested, and established the scoring algorithm for predicting TALEN activity (SAPTA) as a new online design tool. For any input gene of interest, SAPTA gives a ranked list of potential TALEN target sites, facilitating the selection of optimal TALEN pairs based on predicted activity. SAPTA-based TALEN designs increased the average intracellular TALEN monomer activity by >3-fold, and resulted in an average endogenous gene-modification frequency of 39% for TALENs containing the repeat variable di-residue NK that favors specificity rather than activity. It is expected that SAPTA will become a useful and flexible tool for designing highly active TALENs for genome-editing applications. SAPTA can be accessed via the website at http://baolab.bme.gatech.edu/Research/BioinformaticTools/TAL_targeter.html.

  15. Myostatin gene mutated mice induced with tale nucleases.

    PubMed

    Zhou, Fangfang; Sun, Ruilin; Chen, Hongyan; Fei, Jian; Lu, Daru

    2015-01-01

    Myostain gene (MSTN) is expressed primarily in skeletal muscle, and negatively regulates skeletal muscle mass; it has been suggested that mice with MSTN inhibition have reduced adiposity and improved insulin sensitivity. Therefore, it is important to establish a fast and effective gene editing method. In this report, we established the myostatin mutated-mouse model by microinjection of Transcription Activator-Like Effector Nucleases (TALENs) mRNA within the mouse fertilized oocytes and achieved high rates of mutagenesis of the mouse MSTN in C57BL/6J. Six of 45 born mice carried target mutations and we appointed one as the parental mating with wild mouse to produce the F1 and backcross to produce the F2 generation. All the mutations of the mice were examined quickly and efficiently by high-resolution melting curve analysis (HRMA) and then verified by direct sequencing. We obtained the homozygous of the F2 generation which transmitted the mutant alleles to the progeny with 100% efficiency. Mutant mice exhibited increases in muscle mass comparable to those observed in wild-type mice. Therefore, combining TALEN-mediated gene targeting with HRMA technology is a superior method of constructing genetically modified mice through microinjection in the mouse fertilized oocytes with high efficiency and short time of selection.

  16. Double Perspectives: Old Tales in Modern Novels.

    ERIC Educational Resources Information Center

    Perkins, Agnes

    There is a wealth of material in the oral tradition of fantasy which can be used in creating gripping fiction for young readers. One way of employing a tale from the oral tradition to produce a modern fantasy is to superimpose the old story on a new one in such a way that it is tightly interwoven in the action and meaning of the new story. This…

  17. Minimal mimicry: mere effector matching induces preference.

    PubMed

    Sparenberg, Peggy; Topolinski, Sascha; Springer, Anne; Prinz, Wolfgang

    2012-12-01

    Both mimicking and being mimicked induces preference for a target. The present experiments investigate the minimal sufficient conditions for this mimicry-preference link to occur. We argue that mere effector matching between one's own and the other person's movement is sufficient to induce preference, independent of which movement is actually performed. In Experiments 1 and 2, participants moved either their arms or legs, and watched avatars that moved either their arms or legs, respectively, without any instructions to mimic. The executed movements themselves and their pace were completely different between participants (fast circular movements) and targets (slow linear movements). Participants preferred avatars that moved the same body part as they did over avatars that moved a different body part. In Experiment 3, using human targets and differently paced movements, movement similarity was manipulated in addition to effector overlap (moving forward-backward or sideways with arms or legs, respectively). Only effector matching, but not movement matching, influenced preference ratings. These findings suggest that mere effector overlap is sufficient to trigger preference by mimicry.

  18. Cellular effector mechanisms against Plasmodium liver stages.

    PubMed

    Frevert, Ute; Nardin, Elizabeth

    2008-10-01

    Advances in our understanding of the molecular and cell biology of the malaria parasite have led to new vaccine development efforts resulting in a pipeline of over 40 candidates undergoing clinical phase I-III trials. Vaccine-induced CD4+ and CD8+ T cells specific for pre-erythrocytic stage antigens have been found to express cytolytic and multi-cytokine effector functions that support a key role for these T cells within the hepatic environment. However, little is known of the cellular interactions that occur during the effector phase in which the intracellular hepatic stage of the parasite is targeted and destroyed. This review focuses on cell biological aspects of the interaction between malaria-specific effector cells and the various antigen-presenting cells that are known to exist within the liver, including hepatocytes, dendritic cells, Kupffer cells, stellate cells and sinusoidal endothelia. Considering the unique immune properties of the liver, it is conceivable that these different hepatic antigen-presenting cells fulfil distinct but complementary roles during the effector phase against Plasmodium liver stages.

  19. Kinematic evaluation of end effector design

    NASA Astrophysics Data System (ADS)

    Edwards, Gary W.

    1992-09-01

    The complex, many degree-of-freedom end effectors at the leading edge of technology would be unusable in the sea bottom research environment. Simpler designs are required to provide adequate reliability for subsea use. This work examines selection of end effector designs to achieve optimum grasping ability with minimal mechanical complexity. A new method of calculating grasp stability is developed, incorporating elements of previous works in the field. Programs are developed which evaluate the ability of different end effector configurations to grasp representative objects (a cube, sphere, and infinite cylinder). End effector designs considered had circular palms with fingers located at the periphery, oriented so that each pointed to the center of the palm. The program tested configurations of from 1 to 4 fingers and from 1 to 3 links per finger. Three sets of finger proportions were considered: equal length links, half length links, and anthropomorphic proportions. The 2 finger, 2 link per finger configuration was determined to be the optimum design, and the half length proportions were selected as the best set of proportions.

  20. Minimal Mimicry: Mere Effector Matching Induces Preference

    ERIC Educational Resources Information Center

    Sparenberg, Peggy; Topolinski, Sascha; Springer, Anne; Prinz, Wolfgang

    2012-01-01

    Both mimicking and being mimicked induces preference for a target. The present experiments investigate the minimal sufficient conditions for this mimicry-preference link to occur. We argue that mere effector matching between one's own and the other person's movement is sufficient to induce preference, independent of which movement is actually…

  1. Morality's ugly implications in Oscar Wilde's fairy tales.

    PubMed

    Jones, Justin T

    2011-01-01

    In Oscar Wilde's two volumes of fairy tales, "The Happy Prince" and Other Tales (1888) and A House of Pomegranates (1891), many central characters meet with premature death or physical disfigurement after learning a bourgeois moral lesson. In an attempt to explain this unconventional phenomenon in the fairy tale tradition, this essay examines Wilde's stories through the lens of his aesthetic ideology and demonstrates how the superficial morality of the Victorian bourgeoisie corrodes each tale's aesthetic integrity, causing the characters to either deny morality outright, assume the guise of Christian philanthropy, or die as the result of their moral reformation.

  2. A catalogue of the effector secretome of plant pathogenic oomycetes.

    PubMed

    Kamoun, Sophien

    2006-01-01

    The oomycetes form a phylogenetically distinct group of eukaryotic microorganisms that includes some of the most notorious pathogens of plants. Oomycetes accomplish parasitic colonization of plants by modulating host cell defenses through an array of disease effector proteins. The biology of effectors is poorly understood but tremendous progress has been made in recent years. This review classifies and catalogues the effector secretome of oomycetes. Two classes of effectors target distinct sites in the host plant: Apoplastic effectors are secreted into the plant extracellular space, and cytoplasmic effectors are translocated inside the plant cell, where they target different subcellular compartments. Considering that five species are undergoing genome sequencing and annotation, we are rapidly moving toward genome-wide catalogues of oomycete effectors. Already, it is evident that the effector secretome of pathogenic oomycetes is more complex than expected, with perhaps several hundred proteins dedicated to manipulating host cell structure and function.

  3. The RAS-Effector Interface: Isoform-Specific Differences in the Effector Binding Regions

    PubMed Central

    Nakhaeizadeh, Hossein; Amin, Ehsan; Nakhaei-Rad, Saeideh; Dvorsky, Radovan; Ahmadian, Mohammad Reza

    2016-01-01

    RAS effectors specifically interact with the GTP-bound form of RAS in response to extracellular signals and link them to downstream signaling pathways. The molecular nature of effector interaction by RAS is well-studied but yet still incompletely understood in a comprehensive and systematic way. Here, structure-function relationships in the interaction between different RAS proteins and various effectors were investigated in detail by combining our in vitro data with in silico data. Equilibrium dissociation constants were determined for the binding of HRAS, KRAS, NRAS, RRAS1 and RRAS2 to both the RAS binding (RB) domain of CRAF and PI3Kα, and the RAS association (RA) domain of RASSF5, RALGDS and PLCε, respectively, using fluorescence polarization. An interaction matrix, constructed on the basis of available crystal structures, allowed identification of hotspots as critical determinants for RAS-effector interaction. New insights provided by this study are the dissection of the identified hotspots in five distinct regions (R1 to R5) in spite of high sequence variability not only between, but also within, RB/RA domain-containing effectors proteins. Finally, we propose that intermolecular β-sheet interaction in R1 is a central recognition region while R3 may determine specific contacts of RAS versus RRAS isoforms with effectors. PMID:27936046

  4. The broadly effective recessive resistance gene xa5 of rice is a virulence effector-dependent quantitative trait for bacterial blight.

    PubMed

    Huang, Sheng; Antony, Ginny; Li, Ting; Liu, Bo; Obasa, Ken; Yang, Bing; White, Frank F

    2016-04-01

    Mutations in disease susceptibility (S) genes, here referred to as recessive resistance genes, have promise for providing broad durable resistance in crop species. However, few recessive disease resistance genes have been characterized. Here, we show that the broadly effective resistance gene xa5,for resistance to bacterial blight of rice (Oryza sativa), is dependent on the effector genes present in the pathogen. Specifically, the effectiveness of xa5 in preventing disease by strains of Xanthomonas oryzae pv. oryzae is dependent on major transcription activation-like (TAL) effector genes, and correlates with reduced expression of the cognate S genes. xa5 is ineffective in preventing disease by strains containing the TAL effector gene pthXo1, which directs robust expression of the S gene OsSWEET11, a member of sucrose transporter gene family. Incompatibility is associated with major TAL effectors that target the known alternative S genes OsSWEET14 and OsSWEET13. Incompatibility is defeated by transfer of pthXo1 to otherwise xa5-incompatible strains or by engineering a synthetic designer TAL effector to boost SWEET gene expression. In either case, compatible or incompatible, target gene expression and lesion formation are reduced in the presence of xa5. The results indicate that xa5 functions as a quantitative trait locus, dampening effector function, and, regardless of compatibility, target gene expression. Resistance is hypothesized to occur when S gene expression, and, by inference, sucrose leakage, falls below a threshold level.

  5. Spinning New Tales from Traditional Texts: Donna Jo Napoli and the Rewriting of Fairy Tale.

    ERIC Educational Resources Information Center

    Crew, Hilary S.

    2002-01-01

    Demonstrates how Donna Jo Napoli changes generic conventions and reworks discursive formations in order to retell tradition tales. Discusses the narrative strategies she uses in telling her stories, her representation of male and female characters in regard to gender and gendered relationships, and the way she renegotiates ideologies and value…

  6. Identification of putative TAL effector targets of the citrus canker pathogens shows functional convergence underlying disease development and defense response

    PubMed Central

    2014-01-01

    Background Transcriptional activator-like (TAL) effectors, formerly known as the AvrBs3/PthA protein family, are DNA-binding effectors broadly found in Xanthomonas spp. that transactivate host genes upon injection via the bacterial type three-secretion system. Biologically relevant targets of TAL effectors, i.e. host genes whose induction is vital to establish a compatible interaction, have been reported for xanthomonads that colonize rice and pepper; however, citrus genes modulated by the TAL effectors PthA“s” and PthC“s” of the citrus canker bacteria Xanthomonas citri (Xc) and Xanthomonas aurantifolii pathotype C (XaC), respectively, are poorly characterized. Of particular interest, XaC causes canker disease in its host lemon (Citrus aurantifolia), but triggers a defense response in sweet orange. Results Based on, 1) the TAL effector-DNA binding code, 2) gene expression data of Xc and XaC-infiltrated sweet orange leaves, and 3) citrus hypocotyls transformed with PthA2, PthA4 or PthC1, we have identified a collection of Citrus sinensis genes potentially targeted by Xc and XaC TAL effectors. Our results suggest that similar with other strains of Xanthomonas TAL effectors, PthA2 and PthA4, and PthC1 to some extent, functionally converge. In particular, towards induction of genes involved in the auxin and gibberellin synthesis and response, cell division, and defense response. We also present evidence indicating that the TAL effectors act as transcriptional repressors and that the best scoring predicted DNA targets of PthA“s” and PthC“s” in citrus promoters predominantly overlap with or localize near to TATA boxes of core promoters, supporting the idea that TAL effectors interact with the host basal transcriptional machinery to recruit the RNA pol II and start transcription. Conclusions The identification of PthA“s” and PthC“s” targets, such as the LOB (LATERAL ORGAN BOUNDARY) and CCNBS genes that we report here, is key for the understanding

  7. Natural effector T lymphocytes in normal mice.

    PubMed Central

    Pereira, P; Larsson, E L; Forni, L; Bandeira, A; Coutinho, A

    1985-01-01

    The "natural" T-cell activity in normal unimmunized mice was studied. By double-parameter fluorescence-activated cell sorter analysis, it was found that 5-10% of all splenic Lyt-2+ and L3T4+ lymphocytes are large, of which more than half are in mitotic cycle. In contrast with small resting cells of the same phenotype, activated (large) T cells isolated from normal mice are functional effector cells: L3T4+ large cells induce normal B lymphocytes into proliferation and antibody secretion, while large Lyt-2+ cells efficiently suppress B-lymphocyte responses. No effector cell cytolytic activity could be detected among naturally activated T cells. The significance of these findings for the internal activity in the normal immune system is discussed. PMID:2933744

  8. Fairy Tales of the Sea [and] A Guide for Teachers.

    ERIC Educational Resources Information Center

    Cowan, Elizabeth, Comp.; And Others

    Presented are 25 fairy tales from around the world that are related to the sea. Included in the teacher's guide are activities designed to serve as a catalyst to stimulate varied classroom uses for the tales. The curriculum outline provided follows the English Language Arts Curriculum Framework suggested by the Texas Education Agency and covers…

  9. Recovering Native Traditions and Tales for Younger Readers.

    ERIC Educational Resources Information Center

    Stott, Jon C.

    1995-01-01

    Reviews 14 children's books concerned with traditional Native American tales and experiences, written mostly by Native authors and published 1989-93. Includes books on Hiawatha, buffalo, the battle of the Little Bighorn, the Fetterman Fight, and traditional beliefs and values; Cree, Navajo, Chickasaw, and Seneca tales and stories; fictional…

  10. The Indecisive Feminist: Study of Anne Sexton's Revisionist Fairy Tales

    ERIC Educational Resources Information Center

    Mohammed, Nadia Fayidh

    2015-01-01

    Fairy tales to female writers are major resource for their abundant writings, but for the feminist poets since 1960s, they become essential subject matter to often deal with in their literary production. With the motivation to address the conventional tradition of patriarchal society, and re-address the stereotype females inhabiting these tales,…

  11. Teaching Trickster Tales: A Comparison of Instructional Approaches

    ERIC Educational Resources Information Center

    Jarvey, Marya; McKeough, Anne; Pyryt, Michael C.

    2008-01-01

    Trickster tales, with their teachings on how to behave in the world, are a powerful means for transmitting social knowledge and cultural mores to children. In this study we compared two approaches to teaching fourth-grade students to write trickster tales. Although both instructional methods incorporated aspects of the writing process approach,…

  12. In Support of Fairy Tales for Language Instruction.

    ERIC Educational Resources Information Center

    Leisher, Johanna

    1984-01-01

    Describes a technique used to teach German to children whereby the children learn a fairy tale in German (in this case, "Hansel and Gretel") and then present the fairy tale as a play. In this way, the students learn basic vocabulary and expressions, German music, and simple folk dances. (SED)

  13. Larger than Life: Reading and Writing Tall Tales

    ERIC Educational Resources Information Center

    Tunks, Karyn

    2008-01-01

    The genre of tall tales is characterized by fictional, often intentionally ridiculous, stories that provide a reason for or origin of a natural phenomenon. Tall tales are often based on characters who are unusually adept or powerful; they are particularly appealing to children who are cognitively capable of understanding the tongue-in-cheek humor…

  14. The Fantastic Tale for Children: Its Literary and Educational Problems.

    ERIC Educational Resources Information Center

    Klingberg, Goete

    1967-01-01

    The fantastic tale is a genre of children's literature in which magic and reality are found side by side in a superficially plausible story with a definite historical setting. Motifs characteristic of the tale are living toys, strange children, modern witches, space and time displacements, "doors" to the wonderland, the mythical world itself, and…

  15. E-Classical Fairy Tales: Multimedia Builder as a Tool

    ERIC Educational Resources Information Center

    Eteokleous, Nikleia; Ktoridou, Despo; Tsolakidis, Symeon

    2011-01-01

    The study examines pre-service teachers' experiences in delivering a traditional-classical fairy tale using the Multimedia Builder software, in other words an e-fairy tale. A case study approach was employed, collecting qualitative data through classroom observations and focus groups. The results focus on pre-service teachers' reactions, opinions,…

  16. The magic of fairy tales: psychodynamic and developmental perspectives.

    PubMed

    Lubetsky, M J

    1989-01-01

    The telling of fairy tales is one way to elicit a child's inner thoughts and feelings, expose conflicts and frustrations, reduce anxiety, and gain mastery over developmental tasks. This paper will review the meaning, usefulness, and importance of fairy tales by discussing three selections from the psychodynamic and developmental perspectives.

  17. Fairy Tales and Foreign Languages: Ever the Twain Shall Meet

    ERIC Educational Resources Information Center

    Davidheiser, James C.

    2007-01-01

    Fairy tales are not new to foreign language instructors, but on occasion they have been considered wither exoteric or unworthy of class time. Yet today there is a resurgence of interest in fairy tales and a rebirth of their use in the arts, which may serve foreign language instructors. This article presents historical background to inform…

  18. Normativity in Fairy Tales: Scope, Range and Modes of Communication

    ERIC Educational Resources Information Center

    Hohr, Hansjörg

    2013-01-01

    The article studies in three steps how the fairy tale articulates its normative content and what the educational consequence of this kind of communication is. First, the articulation of normativity in fictional literature in general is discussed. Second, the specific mode in which the fairy tale articulates its normativity is studied according to…

  19. Novel Control Effectors for Truss Braced Wing

    NASA Technical Reports Server (NTRS)

    White, Edward V.; Kapania, Rakesh K.; Joshi, Shiv

    2015-01-01

    At cruise flight conditions very high aspect ratio/low sweep truss braced wings (TBW) may be subject to design requirements that distinguish them from more highly swept cantilevered wings. High aspect ratio, short chord length and relative thinness of the airfoil sections all contribute to relatively low wing torsional stiffness. This may lead to aeroelastic issues such as aileron reversal and low flutter margins. In order to counteract these issues, high aspect ratio/low sweep wings may need to carry additional high speed control effectors to operate when outboard ailerons are in reversal and/or must carry additional structural weight to enhance torsional stiffness. The novel control effector evaluated in this study is a variable sweep raked wing tip with an aileron control surface. Forward sweep of the tip allows the aileron to align closely with the torsional axis of the wing and operate in a conventional fashion. Aft sweep of the tip creates a large moment arm from the aileron to the wing torsional axis greatly enhancing aileron reversal. The novelty comes from using this enhanced and controllable aileron reversal effect to provide roll control authority by acting as a servo tab and providing roll control through intentional twist of the wing. In this case the reduced torsional stiffness of the wing becomes an advantage to be exploited. The study results show that the novel control effector concept does provide roll control as described, but only for a restricted class of TBW aircraft configurations. For the configuration studied (long range, dual aisle, Mach 0.85 cruise) the novel control effector provides significant benefits including up to 12% reduction in fuel burn.

  20. Avian Interferons and Their Antiviral Effectors.

    PubMed

    Santhakumar, Diwakar; Rubbenstroth, Dennis; Martinez-Sobrido, Luis; Munir, Muhammad

    2017-01-01

    Interferon (IFN) responses, mediated by a myriad of IFN-stimulated genes (ISGs), are the most profound innate immune responses against viruses. Cumulatively, these IFN effectors establish a multilayered antiviral state to safeguard the host against invading viral pathogens. Considerable genetic and functional characterizations of mammalian IFNs and their effectors have been made, and our understanding on the avian IFNs has started to expand. Similar to mammalian counterparts, three types of IFNs have been genetically characterized in most avian species with available annotated genomes. Intriguingly, chickens are capable of mounting potent innate immune responses upon various stimuli in the absence of essential components of IFN pathways including retinoic acid-inducible gene I, IFN regulatory factor 3 (IRF3), and possibility IRF9. Understanding these unique properties of the chicken IFN system would propose valuable targets for the development of potential therapeutics for a broader range of viruses of both veterinary and zoonotic importance. This review outlines recent developments in the roles of avian IFNs and ISGs against viruses and highlights important areas of research toward our understanding of the antiviral functions of IFN effectors against viral infections in birds.

  1. Avian Interferons and Their Antiviral Effectors

    PubMed Central

    Santhakumar, Diwakar; Rubbenstroth, Dennis; Martinez-Sobrido, Luis; Munir, Muhammad

    2017-01-01

    Interferon (IFN) responses, mediated by a myriad of IFN-stimulated genes (ISGs), are the most profound innate immune responses against viruses. Cumulatively, these IFN effectors establish a multilayered antiviral state to safeguard the host against invading viral pathogens. Considerable genetic and functional characterizations of mammalian IFNs and their effectors have been made, and our understanding on the avian IFNs has started to expand. Similar to mammalian counterparts, three types of IFNs have been genetically characterized in most avian species with available annotated genomes. Intriguingly, chickens are capable of mounting potent innate immune responses upon various stimuli in the absence of essential components of IFN pathways including retinoic acid-inducible gene I, IFN regulatory factor 3 (IRF3), and possibility IRF9. Understanding these unique properties of the chicken IFN system would propose valuable targets for the development of potential therapeutics for a broader range of viruses of both veterinary and zoonotic importance. This review outlines recent developments in the roles of avian IFNs and ISGs against viruses and highlights important areas of research toward our understanding of the antiviral functions of IFN effectors against viral infections in birds. PMID:28197148

  2. Impact of end effector technology on telemanipulation performance

    NASA Technical Reports Server (NTRS)

    Bejczy, A. K.; Szakaly, Z.; Ohm, T.

    1990-01-01

    Generic requirements for end effector design are briefly summarized as derived from generic functional and operational requirements. Included is a brief summary of terms and definitions related to end effector technology. The second part contains a brief overview of end effector technology work as JPL during the past ten years, with emphasis on the evolution of new mechanical, sensing and control capabilities of end effectors. The third and major part is devoted to the description of current end effector technology. The ongoing work addresses mechanical, sensing and control details with emphasis on mechanical ruggedness, increased resolution in sensing, and close electronic and control integration with overall telemanipulator control system.

  3. Legionella Metaeffector Exploits Host Proteasome to Temporally Regulate Cognate Effector

    PubMed Central

    Kubori, Tomoko; Shinzawa, Naoaki; Kanuka, Hirotaka; Nagai, Hiroki

    2010-01-01

    Pathogen-associated secretion systems translocate numerous effector proteins into eukaryotic host cells to coordinate cellular processes important for infection. Spatiotemporal regulation is therefore important for modulating distinct activities of effectors at different stages of infection. Here we provide the first evidence of “metaeffector,” a designation for an effector protein that regulates the function of another effector within the host cell. Legionella LubX protein functions as an E3 ubiquitin ligase that hijacks the host proteasome to specifically target the bacterial effector protein SidH for degradation. Delayed delivery of LubX to the host cytoplasm leads to the shutdown of SidH within the host cells at later stages of infection. This demonstrates a sophisticated level of coevolution between eukaryotic cells and L. pneumophila involving an effector that functions as a key regulator to temporally coordinate the function of a cognate effector protein. PMID:21151961

  4. Identification of off-target cleavage sites of zinc finger nucleases and TAL effector nucleases using predictive models.

    PubMed

    Fine, Eli J; Cradick, Thomas J; Bao, Gang

    2014-01-01

    Using engineered nucleases, such as Zinc Finger Nucleases (ZFNs) or Transcription Activator-Like Effector Nucleases (TALENs), to make targeted genomic modifications has become a common technique to create new model organisms and custom cell lines, and has shown great promise for disease treatment. However, these nucleases have the potential for off-target cleavage that could confound interpretation of experimental results and be detrimental for therapeutic use. Here, we describe a method to test for nuclease cleavage at potential off-target sites predicted by bioinformatics models.

  5. Repeat-containing protein effectors of plant-associated organisms

    PubMed Central

    Mesarich, Carl H.; Bowen, Joanna K.; Hamiaux, Cyril; Templeton, Matthew D.

    2015-01-01

    Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms. PMID:26557126

  6. Germline-transmitted genome editing in Arabidopsis thaliana Using TAL-effector-nucleases.

    PubMed

    Forner, Joachim; Pfeiffer, Anne; Langenecker, Tobias; Manavella, Pablo A; Manavella, Pablo; Lohmann, Jan U

    2015-01-01

    Transcription activator-like effector nucleases (TALENs) are custom-made bi-partite endonucleases that have recently been developed and applied for genome engineering in a wide variety of organisms. However, they have been only scarcely used in plants, especially for germline-modification. Here we report the efficient creation of small, germline-transmitted deletions in Arabidopsis thaliana via TALENs that were delivered by stably integrated transgenes. Using meristem specific promoters to drive expression of two TALEN arms directed at the CLV3 coding sequence, we observed very high phenotype frequencies in the T2 generation. In some instances, full CLV3 loss-of-function was already observed in the T1 generation, suggesting that transgenic delivery of TALENs can cause highly efficient genome modification. In contrast, constitutive TALEN expression in the shoot apical meristem (SAM) did not cause additional phenotypes and genome re-sequencing confirmed little off-target effects, demonstrating exquisite target specificity.

  7. Design and fabrication of an end effector

    NASA Technical Reports Server (NTRS)

    Crossley, F. R. E.; Umholtz, F. G.

    1975-01-01

    The construction is described of a prototype mechanical hand or 'end effector' for use on a remotely controlled robot, but with possible application as a prosthetic device. An analysis of hand motions is reported, from which it is concluded that the two most important manipulations (apart from grasps) are to be able to pick up a tool and draw it into a nested grip against the palm, and to be able to hold a pistol-grip tool such as an electric drill and pull the trigger. A model was tested and found capable of both these operations.

  8. Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases.

    PubMed

    Kasparek, Petr; Krausova, Michaela; Haneckova, Radka; Kriz, Vitezslav; Zbodakova, Olga; Korinek, Vladimir; Sedlacek, Radislav

    2014-11-03

    Gene targeting in mice mainly employs homologous recombination (HR) in embryonic stem (ES) cells. Although it is a standard way for production of genetically modified mice, the procedure is laborious and time-consuming. This study describes targeting of the mouse Rosa26 locus by transcription activator-like effector nucleases (TALENs). We employed TALEN-assisted HR in zygotes to introduce constructs encoding TurboRFP and TagBFP fluorescent proteins into the first intron of the Rosa26 gene, and in this way generated two transgenic mice. We also demonstrated that these Rosa26-specific TALENs exhibit high targeting efficiency superior to that of zinc-finger nucleases (ZFNs) specific for the same targeting sequence. Moreover, we devised a reporter assay to assess TALENs activity and specificity to improve the quality of TALEN-assisted targeting.

  9. Once upon anion: a tale of photodetachment.

    PubMed

    Lineberger, W Carl

    2013-01-01

    This contribution is very much a personal history of a journey through the wonderful world of anion chemistry, and a tale of how advances in laser technologies, theoretical methods, and computational capabilities continuously enabled advances in our understanding. It is a story of the excitement and joy that come from the opportunity to add to the fabric of science, and to do so by working as a group of excited explorers with common goals. The participants in this journey include me, my students and postdoctoral associates, my collaborators, and our many generous colleagues. It all happened, in the words of the Beatles, "with a little help from my friends." Actually, it was so much more than a little help!

  10. Once upon Anion: A Tale of Photodetachment

    NASA Astrophysics Data System (ADS)

    Lineberger, W. Carl

    2013-04-01

    This contribution is very much a personal history of a journey through the wonderful world of anion chemistry, and a tale of how advances in laser technologies, theoretical methods, and computational capabilities continuously enabled advances in our understanding. It is a story of the excitement and joy that come from the opportunity to add to the fabric of science, and to do so by working as a group of excited explorers with common goals. The participants in this journey include me, my students and postdoctoral associates, my collaborators, and our many generous colleagues. It all happened, in the words of the Beatles, “with a little help from my friends.” Actually, it was so much more than a little help!

  11. Modification of Bacterial Effector Proteins Inside Eukaryotic Host Cells

    PubMed Central

    Popa, Crina M.; Tabuchi, Mitsuaki; Valls, Marc

    2016-01-01

    Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside the eukaryotic cells. Here, we summarize the recent reports where effector modifications by the eukaryotic machinery have been described. We restrict our focus on proteins secreted by the type III or type IV systems, excluding other bacterial toxins. We describe the known examples of effectors whose enzymatic activity is triggered by interaction with plant and animal cell factors, including GTPases, E2-Ubiquitin conjugates, cyclophilin and thioredoxins. We focus on the structural interactions with these factors and their influence on effector function. We also review the described examples of host-mediated post-translational effector modifications which are required for proper subcellular location and function. These host-specific covalent modifications include phosphorylation, ubiquitination, SUMOylation, and lipidations such as prenylation, fatty acylation and phospholipid binding. PMID:27489796

  12. Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions.

    PubMed

    Wolters, Manuel; Zobiak, Bernd; Nauth, Theresa; Aepfelbacher, Martin

    2015-10-13

    Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

  13. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes.

    PubMed

    Li, Ting; Huang, Sheng; Zhao, Xuefeng; Wright, David A; Carpenter, Susan; Spalding, Martin H; Weeks, Donald P; Yang, Bing

    2011-08-01

    Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a 'modular assembly' method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs to target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry.

  14. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes

    SciTech Connect

    Li, T; Huang, S; Zhao, XF; Wright, DA; Carpenter, S; Spalding, MH; Weeks, DP; Yang, B

    2011-08-08

    Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a 'modular assembly' method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs to target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry.

  15. Fibre optic sensor on robot end effector for flexible assembly

    SciTech Connect

    Yung, K.L.; Lau, W.S.; Choi, C.K.; Shan, Y.Y.

    1995-12-31

    A fibre optic sensor system was constructed for use on robot end effectors for flexible assembly. The sensor detected the deviations between robot end effector and the workpiece. The signal was fed back to robot controller to shift the end effector until the centre of end effector and the centre of workpiece were aligned at the correct orientation. Then workpiece can be grasped symmetrically. Sensor fusion concept was used to guard against sensor system failure. Fuzzy linguistic variable and control rule concept were introduced in the sensor integration. The experimental setup for the sensor integrated system was shown. The accuracy was also discussed.

  16. Resurrecting the buried self: fairy tales and the analytic encounter.

    PubMed

    Jacobs, Linda

    2011-12-01

    The author uses the lens of myth and fairy tales to examine the narratives generated by the analytic experience. Fairy tales are understood as representing fundamental developmental conflicts, accounting for their enduring power over time. The analytic encounter is seen as an analogue of the fairy tale in which the hidden self, damaged by loss and abandonment, reemerges only through the redemptive power of [an] other's love. Clinical material is presented in which hidden parts of the patient's self are projected into the analyst for safekeeping; these hidden parts resonate with the analyst's own lost, unrealized potential and form an intersubjective experience which the author believes is transformative. The patient's dormant powers emerge in a newly experienced atmosphere of recognition, and in this way, the analytic encounter resembles the fairy tale in providing an identificatory bond and a protective space for the patient's hidden vitality.

  17. Active membrane cholesterol as a physiological effector.

    PubMed

    Lange, Yvonne; Steck, Theodore L

    2016-09-01

    Sterols associate preferentially with plasma membrane sphingolipids and saturated phospholipids to form stoichiometric complexes. Cholesterol in molar excess of the capacity of these polar bilayer lipids has a high accessibility and fugacity; we call this fraction active cholesterol. This review first considers how active cholesterol serves as an upstream regulator of cellular sterol homeostasis. The mechanism appears to utilize the redistribution of active cholesterol down its diffusional gradient to the endoplasmic reticulum and mitochondria, where it binds multiple effectors and directs their feedback activity. We have also reviewed a broad literature in search of a role for active cholesterol (as opposed to bulk cholesterol or lipid domains such as rafts) in the activity of diverse membrane proteins. Several systems provide such evidence, implicating, in particular, caveolin-1, various kinds of ABC-type cholesterol transporters, solute transporters, receptors and ion channels. We suggest that this larger role for active cholesterol warrants close attention and can be tested easily.

  18. Miniature Trailing Edge Effector for Aerodynamic Control

    NASA Technical Reports Server (NTRS)

    Lee, Hak-Tae (Inventor); Bieniawski, Stefan R. (Inventor); Kroo, Ilan M. (Inventor)

    2008-01-01

    Improved miniature trailing edge effectors for aerodynamic control are provided. Three types of devices having aerodynamic housings integrated to the trailing edge of an aerodynamic shape are presented, which vary in details of how the control surface can move. A bucket type device has a control surface which is the back part of a C-shaped member having two arms connected by the back section. The C-shaped section is attached to a housing at the ends of the arms, and is rotatable about an axis parallel to the wing trailing edge to provide up, down and neutral states. A flip-up type device has a control surface which rotates about an axis parallel to the wing trailing edge to provide up, down, neutral and brake states. A rotating type device has a control surface which rotates about an axis parallel to the chord line to provide up, down and neutral states.

  19. Rack Insertion End Effector (RIEE) automation

    NASA Technical Reports Server (NTRS)

    Malladi, Narasimha

    1993-01-01

    NASA is developing a mechanism to manipulate and insert Racks into the Space Station Logistic modules. The mechanism consists of the following: a base with three motorized degrees of freedom, a 3 section motorized boom that goes from 15 to 44 feet in length, and a Rack Insertion End Effector (RIEE) with 5 hand wheels for precise alignment. The robotics section was tasked with the automation of the RIEE unit. In this report, for the automation of the RIEE unit, application of the Perceptics Vision System was conceptually developed to determine the position and orientation of the RIEE relative to the logistic module, and a MathCad program is written to display the needed displacements for precise alignment and final insertion of the Rack. The uniqueness of this report is that the whole report is in fact a MathCad program including text, derivations, and executable equations with example inputs and outputs.

  20. Computational Prediction of Effector Proteins in Fungi: Opportunities and Challenges

    PubMed Central

    Sonah, Humira; Deshmukh, Rupesh K.; Bélanger, Richard R.

    2016-01-01

    Effector proteins are mostly secretory proteins that stimulate plant infection by manipulating the host response. Identifying fungal effector proteins and understanding their function is of great importance in efforts to curb losses to plant diseases. Recent advances in high-throughput sequencing technologies have facilitated the availability of several fungal genomes and 1000s of transcriptomes. As a result, the growing amount of genomic information has provided great opportunities to identify putative effector proteins in different fungal species. There is little consensus over the annotation and functionality of effector proteins, and mostly small secretory proteins are considered as effector proteins, a concept that tends to overestimate the number of proteins involved in a plant–pathogen interaction. With the characterization of Avr genes, criteria for computational prediction of effector proteins are becoming more efficient. There are 100s of tools available for the identification of conserved motifs, signature sequences and structural features in the proteins. Many pipelines and online servers, which combine several tools, are made available to perform genome-wide identification of effector proteins. In this review, available tools and pipelines, their strength and limitations for effective identification of fungal effector proteins are discussed. We also present an exhaustive list of classically secreted proteins along with their key conserved motifs found in 12 common plant pathogens (11 fungi and one oomycete) through an analytical pipeline. PMID:26904083

  1. The Pseudomonas syringae effector protein HopZ1a suppresses effector-triggered immunity.

    PubMed

    Macho, Alberto P; Guevara, Carlos M; Tornero, Pablo; Ruiz-Albert, Javier; Beuzón, Carmen R

    2010-09-01

    *The Pseudomonas syringae pv syringae type III effector HopZ1a is a member of the HopZ effector family of cysteine-proteases that triggers immunity in Arabidopsis. This immunity is dependent on HopZ1a cysteine-protease activity, and independent of known resistance genes. We have previously shown that HopZ1a-triggered immunity is partially additive to that triggered by AvrRpt2. These partially additive effects could be caused by at least two mechanisms: their signalling pathways share a common element(s), or one effector interferes with the response triggered by the other. *Here, we investigate the molecular basis for the partially additive effect displayed by AvrRpt2- and HopZ1a-triggered immunities, by analysing competitive indices, hypersensitive response and symptom induction, PR-1 accumulation, expression of PR genes, and systemic acquired resistance (SAR) induction. *Partially additive effects between these defence responses require HopZ1a cysteine-protease activity, and also take place between HopZ1a and AvrRps4 or AvrRpm1-triggered responses. We establish that HopZ1a-triggered immunity is independent of salicylic acid (SA), EDS1, jasmonic acid (JA) and ethylene (ET)-dependent pathways, and show that HopZ1a suppresses the induction of PR-1 and PR-5 associated with P. syringae pv tomato (Pto)-triggered effector-triggered immunity (ETI)-like defences, AvrRpt2-triggered immunity, and Pto or Pto (avrRpt2) activation of SAR, and that suppression requires HopZ1a cysteine-protease activity. *Our results indicate that HopZ1a triggers an unusual resistance independent of known pathways and suppresses SA and EDS1-dependent resistance.

  2. Double lead spiral platen parallel jaw end effector

    NASA Technical Reports Server (NTRS)

    Beals, David C.

    1989-01-01

    The double lead spiral platen parallel jaw end effector is an extremely powerful, compact, and highly controllable end effector that represents a significant improvement in gripping force and efficiency over the LaRC Puma (LP) end effector. The spiral end effector is very simple in its design and has relatively few parts. The jaw openings are highly predictable and linear, making it an ideal candidate for remote control. The finger speed is within acceptable working limits and can be modified to meet the user needs; for instance, greater finger speed could be obtained by increasing the pitch of the spiral. The force relaxation is comparable to the other tested units. Optimization of the end effector design would involve a compromise of force and speed for a given application.

  3. Secretion, delivery and function of oomycete effector proteins.

    PubMed

    Wawra, Stephan; Belmonte, Rodrigo; Löbach, Lars; Saraiva, Marcia; Willems, Ariane; van West, Pieter

    2012-12-01

    Oomycetes are responsible for multi-billion dollar damages in aquaculture, agriculture and forestry. One common strategy they share with most cellular disease agents is the secretion of effector proteins. Effectors are molecules that change host physiology by initiating and allowing an infection to develop. Oomycetes secrete both extracellular and intracellular effectors. Studying secretion, delivery and function of effectors will hopefully lead to alternative control measures, which is much needed as several chemicals to control plant and animal pathogenic oomycetes cannot be used anymore; due to resistance in the host, or because the control measures have been prohibited as a result of toxicity to the environment and/or consumers. Here the latest findings on oomycete effector secretion, delivery and function are discussed.

  4. Type III effector-mediated processes in Salmonella infection.

    PubMed

    van der Heijden, Joris; Finlay, B Brett

    2012-06-01

    Salmonella is one of the most successful bacterial pathogens that infect humans in both developed and developing countries. In order to cause infection, Salmonella uses type III secretion systems to inject bacterial effector proteins into host cells. In the age of antibiotic resistance, researchers have been looking for new strategies to reduce Salmonella infection. To understand infection and to analyze type III secretion as a potential therapeutic target, research has focused on identification of effectors, characterization of effector functions and how they contribute to disease. Many effector-mediated processes have been identified that contribute to infection but thus far no specific treatment has been found. In this perspective we discuss our current understanding of effector-mediated processes and discuss new techniques and approaches that may help us to find a solution to this worldwide problem.

  5. Advanced Aerodynamic Design of Passive Porosity Control Effectors

    NASA Technical Reports Server (NTRS)

    Hunter, Craig A.; Viken, Sally A.; Wood, Richard M.; Bauer, Steven X. S.

    2001-01-01

    This paper describes aerodynamic design work aimed at developing a passive porosity control effector system for a generic tailless fighter aircraft. As part of this work, a computational design tool was developed and used to layout passive porosity effector systems for longitudinal and lateral-directional control at a low-speed, high angle of attack condition. Aerodynamic analysis was conducted using the NASA Langley computational fluid dynamics code USM3D, in conjunction with a newly formulated surface boundary condition for passive porosity. Results indicate that passive porosity effectors can provide maneuver control increments that equal and exceed those of conventional aerodynamic effectors for low-speed, high-alpha flight, with control levels that are a linear function of porous area. This work demonstrates the tremendous potential of passive porosity to yield simple control effector systems that have no external moving parts and will preserve an aircraft's fixed outer mold line.

  6. Action selection in multi-effector decision making

    PubMed Central

    Madlon-Kay, Seth; Pesaran, Bijan; Daw, Nathaniel D.

    2012-01-01

    Decision making and reinforcement learning over movements suffer from the curse of dimensionality: the space of possible movements is too vast to search or even represent in its entirety. When actions involve only a single effector, this problem can be ameliorated by considering that effector separately; accordingly, the brain’s sensorimotor systems can subdivide choice by representing values and actions separately for each effector. However, for many actions, such as playing the piano, the value of an action by an effector (e.g., a hand) depends inseparably on the actions of other effectors. By definition, the values of such coordinated multi-effector actions cannot be represented by effector-specific action values, such as those that have been most extensively investigated in parietal and premotor regions. For such actions, one possible solution is to choose according to more abstract valuations over different goods or options, which can then be mapped onto the necessary motor actions. Such an approach separates the learning and decision problem, which will often be lower-dimensional than the space of possible movements, from the multi-effector movement planning problem. The ventromedial prefrontal cortex (vmPFC) is thought to contain goods-based value signals, so we hypothesized that this region might preferentially drive multi-effector action selection. To examine how the brain solves this problem, we used fMRI to compare patterns of BOLD activity in humans during reward learning tasks in which options were selected through either unimanual or bimanual actions, and in which the response requirements in the latter condition inseparably coupled valuation across both hands. We found value signals in the bilateral medial motor cortex and vmPFC, and consistent with previous studies, the medial motor value signals contained contra-lateral biases indicating effector-specificity, while the vmPFC value signals did not exhibit detectable effector specificity. Although

  7. Signals for the execution of Th2 effector function.

    PubMed

    Fowell, Deborah J

    2009-04-01

    Appropriate control of infection depends on the generation of lymphocytes armed with a particular array of cytokine and chemokine effector molecules. The differentiation of naïve T cells into functionally distinct effector subsets is regulated by signals from the T cell receptor (TCR) and cytokine receptors. Using gene knock-out approaches, the initiation of discrete effector programs appears differentially sensitive to the loss of individual TCR signaling components; likely due to differences in the transcription factors needed to activate individual cytokine genes. Less well understood however, are the signal requirements for the execution of effector function. With a focus on Th2 cells and the kinase ITK, we review recent observations that point to differences between the signals needed for the initiation and implementation of cytokine programs in CD4+ T cells. Indeed, Th2 effector cells signal differently from both their naïve counterparts and from Th1 effectors suggesting they may transduce activation signals differently or may be selectively receptive to different activation signals. Potential regulation points for effector function lie at the level of transcription and translation of cytokine genes. We also discuss how provision of these execution signals may be spatially segregated in vivo occurring at tissue sites of inflammation and subject to modulation by the pathogen itself.

  8. Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice.

    PubMed

    Zhou, Junhui; Peng, Zhao; Long, Juying; Sosso, Davide; Liu, Bo; Eom, Joon-Seob; Huang, Sheng; Liu, Sanzhen; Vera Cruz, Casiana; Frommer, Wolf B; White, Frank F; Yang, Bing

    2015-05-01

    Bacterial blight of rice is caused by the γ-proteobacterium Xanthomonas oryzae pv. oryzae, which utilizes a group of type III TAL (transcription activator-like) effectors to induce host gene expression and condition host susceptibility. Five SWEET genes are functionally redundant to support bacterial disease, but only two were experimentally proven targets of natural TAL effectors. Here, we report the identification of the sucrose transporter gene OsSWEET13 as the disease-susceptibility gene for PthXo2 and the existence of cryptic recessive resistance to PthXo2-dependent X. oryzae pv. oryzae due to promoter variations of OsSWEET13 in japonica rice. PthXo2-containing strains induce OsSWEET13 in indica rice IR24 due to the presence of an unpredicted and undescribed effector binding site not present in the alleles in japonica rice Nipponbare and Kitaake. The specificity of effector-associated gene induction and disease susceptibility is attributable to a single nucleotide polymorphism (SNP), which is also found in a polymorphic allele of OsSWEET13 known as the recessive resistance gene xa25 from the rice cultivar Minghui 63. The mutation of OsSWEET13 with CRISPR/Cas9 technology further corroborates the requirement of OsSWEET13 expression for the state of PthXo2-dependent disease susceptibility to X. oryzae pv. oryzae. Gene profiling of a collection of 104 strains revealed OsSWEET13 induction by 42 isolates of X. oryzae pv. oryzae. Heterologous expression of OsSWEET13 in Nicotiana benthamiana leaf cells elevates sucrose concentrations in the apoplasm. The results corroborate a model whereby X. oryzae pv. oryzae enhances the release of sucrose from host cells in order to exploit the host resources.

  9. Computational investigation of miniature trailing edge effectors

    NASA Astrophysics Data System (ADS)

    Lee, Hak-Tae

    Miniature trailing edge effectors (MiTEs) are small flaps (typically 1% to 5% chord) actuated with deflection angles of up to 90 degrees. The small size, combined with little required power and good control authority, enables the device to be used for high bandwidth control as well as conventional attitude control. However, some of the aerodynamic characteristics of these devices are complex and poorly understood. This research investigated the aerodynamics of MiTEs using incompressible Navier-Stokes flow solvers, INS2D and INS3D. To understand the flow structure and establish a parametric database, two dimensional steady-state computations were performed for MiTEs with various geometries and flow conditions. Time accurate computations were used to resolve the unsteady characteristics including transient response and vortex shedding phenomena. The frequency response was studied to fully identify the dynamics of MiTEs. Three dimensional computations show the change in control effectiveness with respect to the spanwise length of MiTEs as well as the spanwise lift distribution induced by these devices. Based on the CFD results, an approximate vortex panel model was developed for design purposes that reproduces the key characteristics of MiTEs. Two application areas for MiTEs were explored. Flutter suppression was demonstrated by combining a finite element structural model with the vortex panel model. The application of MiTEs to augment maximum lift and improve the post stall behavior of an airfoil was also investigated.

  10. Bioprospecting open reading frames for peptide effectors.

    PubMed

    Xiong, Ling; Scott, Charles

    2014-01-01

    Recent successes in the development of small-molecule antagonists of protein-protein interactions designed based on co-crystal structures of peptides bound to their biological targets confirm that short peptides derived from interacting proteins can be high-value ligands for pharmacologic validation of targets and for identification of druggable sites. Evolved sequence space is likely to be enriched for interacting peptides, but identifying minimal peptide effectors within genomic sequence can be labor intensive. Here we describe the use of incremental truncation to diversify genetic material on the scale of open reading frames into comprehensive libraries of constituent peptides. The approach is capable of generating peptides derived from both continuous and discontinuous sequence elements, and is compatible with the expression of free linear or backbone cyclic peptides, with peptides tethered to amino- or carboxyl-terminal fusion partners or with the expression of peptides displayed within protein scaffolds (peptide aptamers). Incremental truncation affords a valuable source of molecular diversity to interrogate the druggable genome or evaluate the therapeutic potential of candidate genes.

  11. Rack Insertion End Effector (RIEE) guidance

    NASA Technical Reports Server (NTRS)

    Malladi, Narasimha S.

    1994-01-01

    NASA-KSC has developed a mechanism to handle and insert Racks into the Space Station Logistic Modules. This mechanism consists of a Base with 3 motorized degrees of freedom, a 3 section motorized Boom that goes from 15 to 44 feet in length, and a Rack Insertion End Effector (RIEE) with 5 hand wheels for precise alignment. During the 1993 NASA-ASEE Summer Faculty Fellowship Program at KSC, I designed an Active Vision (Camera) Arrangement and developed an algorithm to determine (1) the displacements required by the Room for its initial positioning and (2) the rotations required at the five hand-wheels of the RIEE, for the insertion of the Rack, using the centroids fo the Camera Images of the Location Targets in the Logistic Module. Presently, during the summer of '94, I completed the preliminary design of an easily portable measuring instrument using encoders to obtain the 3-Dimensional Coordinates of Location Targets in the Logistics Module relative to the RIEE mechanism frame. The algorithm developed in '93 can use the output of this instrument also. Simplification of the '93 work and suggestions for the future work are discussed.

  12. Effectors from Wheat Rust Fungi Suppress Multiple Plant Defense Responses.

    PubMed

    Ramachandran, Sowmya R; Yin, Chuntao; Kud, Joanna; Tanaka, Kiwamu; Mahoney, Aaron K; Xiao, Fangming; Hulbert, Scot H

    2017-01-01

    Fungi that cause cereal rust diseases (genus Puccinia) are important pathogens of wheat globally. Upon infection, the fungus secretes a number of effector proteins. Although a large repository of putative effectors has been predicted using bioinformatic pipelines, the lack of available high-throughput effector screening systems has limited functional studies on these proteins. In this study, we mined the available transcriptomes of Puccinia graminis and P. striiformis to look for potential effectors that suppress host hypersensitive response (HR). Twenty small (<300 amino acids), secreted proteins, with no predicted functions were selected for the HR suppression assay using Nicotiana benthamiana, in which each of the proteins were transiently expressed and evaluated for their ability to suppress HR caused by four cytotoxic effector-R gene combinations (Cp/Rx, ATR13/RPP13, Rpt2/RPS-2, and GPA/RBP-1) and one mutated R gene-Pto(Y207D). Nine out of twenty proteins, designated Shr1 to Shr9 (suppressors of hypersensitive response), were found to suppress HR in N. benthamiana. These effectors varied in the effector-R gene defenses they suppressed, indicating these pathogens can interfere with a variety of host defense pathways. In addition to HR suppression, effector Shr7 also suppressed PAMP-triggered immune response triggered by flg22. Finally, delivery of Shr7 through Pseudomonas fluorescens EtHAn suppressed nonspecific HR induced by Pseudomonas syringae DC3000 in wheat, confirming its activity in a homologous system. Overall, this study provides the first evidence for the presence of effectors in Puccinia species suppressing multiple plant defense responses.

  13. A tale of two acute extradural hematomas

    PubMed Central

    Adeleye, Amos Olufemi; Jite, Ikechi E.; Smith, Omolara A.

    2016-01-01

    Background: In much of the Western hemisphere, mortality from traumatic acute extradural hematomas (AEDH) has been drastically brought down toward 0%. This is still not the case however in most developing countries. Case Description: This report represents a tragi-comic tale of two cases of traumatic AEDH managed by an academic neurosurgeon in a neurosurgically ill-resourced private health facility during a nationwide industrial strike action preventing clinical-surgical care in the principal author's University Teaching Hospital. A young man presented with altered consciousness, Glasgow Coma Score (GCS) 14/15, following a road accident. The cranial computed tomography (CT) scan was obtained only 9 h after its request, long after the man had actually deteriorated to GCS 7/15 with pupillary changes. The neurosurgeon, summoned from the nearby University Teaching Hospital for the operative care of this man, arrived on-site and was about moving the patient into the operative room when he took the final breaths and died, all within 2 h of the belated neuroimaging. This scenario repeated itself in the same health facility just 24 h later with another young man who presented GCS 7/15 and another identical CT evidence of traumatic AEDH. With more financially able relations, the diagnostic/surgical care of this second patient was much more prompt. He made a very brisk recovery from neurosurgical operative intervention. He is alive and well, 5-month postoperative. Conclusions: In most low-resourced health systems of the developing countries, a significant proportion of potentially salvageable cases of AEDH still perish from this disease condition. PMID:27213108

  14. The TAL effector PthA4 interacts with nuclear factors involved in RNA-dependent processes including a HMG protein that selectively binds poly(U) RNA.

    PubMed

    de Souza, Tiago Antonio; Soprano, Adriana Santos; de Lira, Nayara Patricia Vieira; Quaresma, Alexandre José Christino; Pauletti, Bianca Alves; Paes Leme, Adriana Franco; Benedetti, Celso Eduardo

    2012-01-01

    Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL) effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated with transcription control and DNA repair. To extend our knowledge on the mode of action of PthAs, we have identified new protein targets of the PthA4 variant, required to elicit canker on citrus. Here we show that all the PthA4-interacting proteins are DNA and/or RNA-binding factors implicated in chromatin remodeling and repair, gene regulation and mRNA stabilization/modification. The majority of these proteins, including a structural maintenance of chromosomes protein (CsSMC), a translin-associated factor X (CsTRAX), a VirE2-interacting protein (CsVIP2), a high mobility group (CsHMG) and two poly(A)-binding proteins (CsPABP1 and 2), interacted with each other, suggesting that they assemble into a multiprotein complex. CsHMG was shown to bind DNA and to interact with the invariable leucine-rich repeat region of PthAs. Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U) RNA, a property that is novel among HMGs and TAL effectors. Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control.

  15. System for exchanging tools and end effectors on a robot

    DOEpatents

    Burry, D.B.; Williams, P.M.

    1991-02-19

    A system and method for exchanging tools and end effectors on a robot permits exchange during a programmed task. The exchange mechanism is located off the robot, thus reducing the mass of the robot arm and permitting smaller robots to perform designated tasks. A simple spring/collet mechanism mounted on the robot is used which permits the engagement and disengagement of the tool or end effector without the need for a rotational orientation of the tool to the end effector/collet interface. As the tool changing system is not located on the robot arm no umbilical cords are located on robot. 12 figures.

  16. System for exchanging tools and end effectors on a robot

    DOEpatents

    Burry, David B.; Williams, Paul M.

    1991-02-19

    A system and method for exchanging tools and end effectors on a robot permits exchange during a programmed task. The exchange mechanism is located off the robot, thus reducing the mass of the robot arm and permitting smaller robots to perform designated tasks. A simple spring/collet mechanism mounted on the robot is used which permits the engagement and disengagement of the tool or end effector without the need for a rotational orientation of the tool to the end effector/collet interface. As the tool changing system is not located on the robot arm no umbilical cords are located on robot.

  17. Writing and Retelling Multiple Ethnographic Tales of a Soup Kitchen for the Homeless.

    ERIC Educational Resources Information Center

    Miller, Dana L.; Creswell, John W.; Olander, Lisa

    An ethnographic study narrated three tales about a soup kitchen for the homeless and the near-homeless. To provide a cultural, ethnographic analysis, and share fieldwork experiences the study began with realist and confessional tales. These two tales emerged from the initial writing and presenting of the soup kitchen ethnography to qualitative…

  18. The Fantastic Tale for Children: A Genre Study from the Viewpoints of Literary and Educational Research.

    ERIC Educational Resources Information Center

    Klingberg, Gote

    The literary genre explored in this study is The Fantastic Tale for Children, which combines magic and reality. Two examples of the "pure" fantastic tale are "The Return of the Twelves" by Pauline Clarke and "Tom's Midnight Garden" by Philippa Pearce. The motifs of the fantastic tale are methods which connect the world of the principal characters…

  19. Ugiuvangmiut Quliapyuit = King Island Tales. Eskimo History and Legends from Bering Strait.

    ERIC Educational Resources Information Center

    Kaplan, Lawrence D., Ed.

    The collection of native tales from King Island, Alaska, contains tales told originally in Inupiaq Eskimo by seven native elders. Introductory sections provide background information on the storytellers, King Island Village and its people, traditional life there, and the language of the King Islanders. The 25 tales are divided into groups:…

  20. Actin dynamics shape microglia effector functions.

    PubMed

    Uhlemann, Ria; Gertz, Karen; Boehmerle, Wolfgang; Schwarz, Tobias; Nolte, Christiane; Freyer, Dorette; Kettenmann, Helmut; Endres, Matthias; Kronenberg, Golo

    2016-06-01

    Impaired actin filament dynamics have been associated with cellular senescence. Microglia, the resident immune cells of the brain, are emerging as a central pathophysiological player in neurodegeneration. Microglia activation, which ranges on a continuum between classical and alternative, may be of critical importance to brain disease. Using genetic and pharmacological manipulations, we studied the effects of alterations in actin dynamics on microglia effector functions. Disruption of actin dynamics did not affect transcription of genes involved in the LPS-triggered classical inflammatory response. By contrast, in consequence of impaired nuclear translocation of phospho-STAT6, genes involved in IL-4 induced alternative activation were strongly downregulated. Functionally, impaired actin dynamics resulted in reduced NO secretion and reduced release of TNFalpha and IL-6 from LPS-stimulated microglia and of IGF-1 from IL-4 stimulated microglia. However, pathological stabilization of the actin cytoskeleton increased LPS-induced release of IL-1beta and IL-18, which belong to an unconventional secretory pathway. Reduced NO release was associated with decreased cytoplasmic iNOS protein expression and decreased intracellular arginine uptake. Furthermore, disruption of actin dynamics resulted in reduced microglia migration, proliferation and phagocytosis. Finally, baseline and ATP-induced [Ca(2+)]int levels were significantly increased in microglia lacking gelsolin, a key actin-severing protein. Together, the dynamic state of the actin cytoskeleton profoundly and distinctly affects microglia behaviours. Disruption of actin dynamics attenuates M2 polarization by inhibiting transcription of alternative activation genes. In classical activation, the role of actin remodelling is complex, does not relate to gene transcription and shows a major divergence between cytokines following conventional and unconventional secretion.

  1. Genomic analysis of 38 Legionella species identifies large and diverse effector repertoires.

    PubMed

    Burstein, David; Amaro, Francisco; Zusman, Tal; Lifshitz, Ziv; Cohen, Ofir; Gilbert, Jack A; Pupko, Tal; Shuman, Howard A; Segal, Gil

    2016-02-01

    Infection by the human pathogen Legionella pneumophila relies on the translocation of ∼ 300 virulence proteins, termed effectors, which manipulate host cell processes. However, almost no information exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species and predicted their effector repertoires using a previously validated machine learning approach. This analysis identified 5,885 predicted effectors. The effector repertoires of different Legionella species were found to be largely non-overlapping, and only seven core effectors were shared by all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from the natural protozoan hosts of these species. Furthermore, we detected numerous new conserved effector domains and discovered new domain combinations, which allowed the inference of as yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution.

  2. An intelligent end-effector for a rehabilitation robot.

    PubMed

    Gosine, R G; Harwin, W S; Furby, L J; Jackson, R D

    1989-01-01

    A UMI RTX robot, modified with limited end-effector sensors and a restricted but effective vision system, is currently used in a developmental education setting for severely physically disabled children. The low physical and cognitive abilities of the children involved in the project require a semi-autonomous robot with environmental sensing capability to operate in a task oriented mode. A variety of low-cost sensors including proximity, distance, force and slip sensors, have been investigated for integration in end-effectors for the RTX robot. The sensors employed on a modified end-effector are detailed and experimental results are presented. A design for an end-effector with integrated sensors is discussed. The integration of the sensor information into a high-level, task-oriented programming language is detailed and examples of high-level control sequences using sensor inputs are presented. Finally, the development of intelligent gripping strategies based on sensor information is discussed.

  3. Vision-based end-effector position error compensation

    NASA Technical Reports Server (NTRS)

    Bajracharya, Max; Backes, Paul; DiCicco, Matthew

    2006-01-01

    This paper describes a computationally efficient algorithm that provides the ability to accurately place an arm end-effector on a target designated in an image using low speed feed back from a fixed stero camera.

  4. Effector-triggered immunity: from pathogen perception to robust defense.

    PubMed

    Cui, Haitao; Tsuda, Kenichi; Parker, Jane E

    2015-01-01

    In plant innate immunity, individual cells have the capacity to sense and respond to pathogen attack. Intracellular recognition mechanisms have evolved to intercept perturbations by pathogen virulence factors (effectors) early in host infection and convert it to rapid defense. One key to resistance success is a polymorphic family of intracellular nucleotide-binding/leucine-rich-repeat (NLR) receptors that detect effector interference in different parts of the cell. Effector-activated NLRs connect, in various ways, to a conserved basal resistance network in order to transcriptionally boost defense programs. Effector-triggered immunity displays remarkable robustness against pathogen disturbance, in part by employing compensatory mechanisms within the defense network. Also, the mobility of some NLRs and coordination of resistance pathways across cell compartments provides flexibility to fine-tune immune outputs. Furthermore, a number of NLRs function close to the nuclear chromatin by balancing actions of defense-repressing and defense-activating transcription factors to program cells dynamically for effective disease resistance.

  5. Gunite Scarifying End Effector. Innovative Technology Summary Report

    SciTech Connect

    2001-09-01

    The Gunite Scarifying End Effector (GSEE) is designed to remove a layer of the gunite tank walls, which are contaminated with radioactivity. Removing this radioactivity is necessary to close the tank.

  6. Interactions of legionella effector proteins with host phosphoinositide lipids.

    PubMed

    Weber, Stephen; Dolinsky, Stephanie; Hilbi, Hubert

    2013-01-01

    By means of the Icm/Dot type IV secretion system Legionella pneumophila translocates several effector proteins into host cells, where they anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. Thus, phosphatidylinositol-4-phosphate anchors the effector proteins SidC and SidM, which promote the interaction of LCVs with the ER and the secretory vesicle trafficking -pathway. In this chapter, we describe protocols to (1) identify PI-binding proteins in Legionella lysates using PI-beads, (2) determine PI-binding specificities and affinities of recombinant Legionella effector proteins by protein-lipid overlays, and (3) use Legionella effectors to identify cellular PI lipids.

  7. Functional redundancy of necrotrophic effectors - consequences for exploitation for breeding.

    PubMed

    Tan, Kar-Chun; Phan, Huyen T T; Rybak, Kasia; John, Evan; Chooi, Yit H; Solomon, Peter S; Oliver, Richard P

    2015-01-01

    Necrotrophic diseases of wheat cause major losses in most wheat growing areas of world. Tan spot (caused by Pyrenophora tritici-repentis) and septoria nodorum blotch (SNB; Parastagonospora nodorum) have been shown to reduce yields by 10-20% across entire agri-ecological zones despite the application of fungicides and a heavy focus over the last 30 years on resistance breeding. Efforts by breeders to improve the resistance of cultivars has been compromised by the universal finding that resistance was quantitative and governed by multiple quantitative trait loci (QTL). Most QTL had a limited effect that was hard to measure precisely and varied significantly from site to site and season to season. The discovery of necrotrophic effectors has given breeding for disease resistance new methods and tools. In the case of tan spot in West Australia, a single effector, PtrToxA and its recogniser gene Tsn1, has a dominating impact in disease resistance. The delivery of ToxA to breeders has had a major impact on cultivar choice and breeding strategies. For P. nodorum, three effectors - SnToxA, SnTox1, and SnTox3 - have been well characterized. Unlike tan spot, no one effector has a dominating role. Genetic analysis of various mapping populations and pathogen isolates has shown that different effectors have varying impact and that epistatic interactions also occur. As a result of these factors the deployment of these effectors for SNB resistance breeding is more complex. We have deleted the three effectors in a strain of P. nodorum and measured effector activity and disease potential of the triple knockout mutant. The culture filtrate causes necrosis in several cultivars and the strain causes disease, albeit the overall levels are less than in the wild type. Modeling of the field disease resistance scores of cultivars from their reactions to the microbially expressed effectors SnToxA, SnTox1, and SnTox3 is significantly improved by including the response to the triple knockout

  8. Design, testing and evaluation of latching end effector

    NASA Technical Reports Server (NTRS)

    Walker, B.; Vandersluis, R.

    1995-01-01

    The Latching End Effector (LEE) forms part of the Space Station Remote Manipulator System (SSRMS) for which Spar Aerospace Ltd, Space Systems Division is the prime contractor. The design, testing and performance evaluation of the Latching End Effector mechanisms is the subject of this paper focusing on: (1) ambient, thermal and vibration testing; (2) snare/rigidize performance testing and interaction during payload acquisition; and (3) latch/umbilical test results and performance.

  9. Views of the manipulator development facility end effector simulation

    NASA Technical Reports Server (NTRS)

    1985-01-01

    Closeup view of the manipulator development facility (MDF) during an end effector simulation showing the two specially made extensions deployed on April 16 by the STS 51-D crewmembers (30953); Medium closeup view of MDF. The white cylindar at right represents the Syncon IV (LEASAT) satellite. The robot device in the foreground is the forearm and end effector of a training version of the remote manipulator system (RMS). Attached to the arm's end are two flyswatter-like tools (30954-5).

  10. Functional heterogeneity of human effector CD8+ T cells.

    PubMed

    Takata, Hiroshi; Naruto, Takuya; Takiguchi, Masafumi

    2012-02-09

    Effector CD8(+) T cells are believed to be terminally differentiated cells having cytotoxic activity and the ability to produce effector cytokines such as INF-γ and TNF-α. We investigated the difference between CXCR1(+) and CXCR1(-) subsets of human effector CD27(-)CD28(-)CD8(+) T cells. The subsets expressed cytolytic molecules similarly and exerted substantial cytolytic activity, whereas only the CXCR1(-) subset had IL-2 productivity and self-proliferative activity and was more resistant to cell death than the CXCR1(+) subset. These differences were explained by the specific up-regulation of CAMK4, SPRY2, and IL-7R in the CXCR1(-) subset and that of pro-apoptotic death-associated protein kinase 1 (DAPK1) in the CXCR1(+) subset. The IL-2 producers were more frequently found in the IL-7R(+) subset of the CXCR1(-) effector CD8(+) T cells than in the IL-7R(-) subset. IL-7/IL-7R signaling promoted cell survival only in the CXCR1(-) subset. The present study has highlighted a novel subset of effector CD8(+) T cells producing IL-2 and suggests the importance of this subset in the homeostasis of effector CD8(+) T cells.

  11. Characterization of the Largest Effector Gene Cluster of Ustilago maydis

    PubMed Central

    Vincon, Volker; Kahmann, Regine

    2014-01-01

    In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function. PMID:24992561

  12. Indian Tales of the Northern Rockies. Indian Culture Series.

    ERIC Educational Resources Information Center

    Old Coyote, Sally; Toineeta, Joy Yellowtail

    Part of the Montana Council for Indian Education's Indian Culture Series, the book contains six folk stories recorded on reservations and by headstart teachers. The stories are: "The Owl", a Gros Ventre tale; "How the Robin Got a Red Breast", from the Flathead Tribe; "Old Man Coyote and the Wild Geese", a Crow Indian…

  13. The Lobster Tale: An Exercise in Critical Thinking

    ERIC Educational Resources Information Center

    Stepanovich, Paul L.

    2009-01-01

    Professors in management and business are encouraged to incorporate critical thinking as an objective in their courses. "The Lobster Tale" provides an opportunity to engage students in various levels of critical thinking, ranging from a relatively superficial reading to an examination of the deeper, often hidden issues. Using the foundations of…

  14. Teaching Tales and Theories: An Ethnographic Next Step?

    ERIC Educational Resources Information Center

    Smith, Louis M.

    A number of methodological and substantive issues were raised by the ethnographic research for the book (coauthored by the author of this paper), "Teaching Tales and Theories: A Story and A Commentary on a Science Classroom" (1981). First, the origin of the "problem" came not only from earlier research but also from a serendipitous meeting with…

  15. Shamans and Kushtakas: North Coast Tales of the Supernatural.

    ERIC Educational Resources Information Center

    Beck, Mary Giraudo

    The Tlingit and Haida are Native Americans who inhabit southeast Alaska and share many traditions and stories. Written by a non-native scholar, this book contains nine Tlingit and Haida tales concerned with shamans and kushtakas. Land otters were fearful hybrid beings of the spirit world. Able to live on land and in water, they had the special…

  16. "Can Drama, through Icelandic Tales, Increase Children's Vocabulary"?

    ERIC Educational Resources Information Center

    Thorkelsdóttir, Rannveig Björk; Ragnarsdóttir, Ása Helga

    2013-01-01

    The article is based on a study, done by Ása Helga Ragnarsdóttir and Rannveig Björk Þorkelsdóttir in 2010-2011 were the authors explored the research question: Can drama, through Icelandic tales, increase children's vocabulary? Methodology of the study was quantitative approach (comparative research). Data was gathered through questionnaires and a…

  17. Aesop and Company: Using Traditional Tales in EFL Classes.

    ERIC Educational Resources Information Center

    Allen, Virginia French

    Making repeated use of a traditional tale can offer various kinds of language practice. Many new teachers use a reading passage just once, investing considerable time in the explanation of the vocabulary needed to understand it, and then rush on to something new. Actually, the best potentialities of the material are still to be tapped, through…

  18. The Tale of Red Emmy: An Irish Witch in Appalachia.

    ERIC Educational Resources Information Center

    Wagaman, Gena D.

    The Appalachian "Tale of Red Emmy" presented in the novel "Oral History" by Lee Smith (1983), reveals both an Irish origin and an American transformation. Granny Younger, one of Smith's narrators, tells of a curse visited on four generations of the Cantrell family after Almarine Cantrell chanced upon the witch Red Emmy in the…

  19. Once upon a Tale. 1995 Florida Library Youth Program.

    ERIC Educational Resources Information Center

    Abramoff, Carolann Palm, Comp.; And Others

    The Florida Library Youth Program is an extension of the Florida Summer Library Program. Many libraries have wanted to provide programs for school-age children at times other than the traditional summer vacation, and this guide responds to their needs. The theme, "Once Upon a Tale," focuses on folklore, stories, and storytelling. The…

  20. Computational Drafting of Plot Structures for Russian Folk Tales.

    PubMed

    Gervás, Pablo

    The plots of stories are known to follow general patterns in terms of their overall structure. This was the basic tenet of structuralist approaches to narratology. Vladimir Propp proposed a procedure for the generation of new tales based on his semi-formal description of the structure of Russian folk tales. This is one of the first existing instances of a creative process described procedurally. The present paper revisits Propp's morphology to build a system that generates instances of Russian folk tales. Propp's view of the folk tale as a rigid sequence of character functions is employed as a plot driver, and some issues that Propp declared relevant but did not explore in detail-such as long-range dependencies between functions or the importance of endings-are given computational shape in the context of a broader architecture that captures all the aspects discussed by Propp. A set of simple evaluation metrics for the resulting outputs is defined inspired on Propp's formalism. The potential of the resulting system for providing a creative story generation system is discussed, and possible lines of future work are discussed.

  1. Construct Validation of the Fairy Tale Test--Standardization Data.

    ERIC Educational Resources Information Center

    Coulacoglou, Carina

    2002-01-01

    Studied the construct validity of the Fairy Tale Test (C. Coulacoglu, 1993), a personality projective test for children, in a sample of 800 Greek children aged 8, 10, and 12. Factor analysis led to identification of eight primary factors, and correlations with other measures provide construct validity evidence. (SLD)

  2. The rise of the undead:Pseudokinases as mediators of effector-triggered immunity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pathogens use effector proteins to suppress host immunity and promote infection. However, plants can recognize specific effectors and mount an effector-triggered immune response that suppresses pathogen growth. The YopJ/HopZ family of type III secreted effector proteins is broadly distributed in bac...

  3. TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants.

    PubMed

    Wendt, Toni; Holm, Preben Bach; Starker, Colby G; Christian, Michelle; Voytas, Daniel F; Brinch-Pedersen, Henrik; Holme, Inger Bæksted

    2013-10-01

    Transcription activator-like effector nucleases (TALENs) enable targeted mutagenesis in a variety of organisms. The primary advantage of TALENs over other sequence-specific nucleases, namely zinc finger nucleases and meganucleases, lies in their ease of assembly, reliability of function, and their broad targeting range. Here we report the assembly of several TALENs for a specific genomic locus in barley. The cleavage activity of individual TALENs was first tested in vivo using a yeast-based, single-strand annealing assay. The most efficient TALEN was then selected for barley transformation. Analysis of the resulting transformants showed that TALEN-induced double strand breaks led to the introduction of short deletions at the target site. Additional analysis revealed that each barley transformant contained a range of different mutations, indicating that mutations occurred independently in different cells.

  4. Fairy Tales and Fantasy: Bridges to Literature--Using Fairy Tales and Fantasy to Motivate the Reluctant Reader.

    ERIC Educational Resources Information Center

    Dungey, Joan M.

    Designed to give children a sense of the cultural heritage that fairy tales represent, this instructional unit was originally developed to motivate eighth-grade low-level readers and was later adapted for English as a second language classes and for a variety of elementary and secondary school learning levels. Objectives of the unit are to help…

  5. Target selection biases from recent experience transfer across effectors.

    PubMed

    Moher, Jeff; Song, Joo-Hyun

    2016-02-01

    Target selection is often biased by an observer's recent experiences. However, not much is known about whether these selection biases influence behavior across different effectors. For example, does looking at a red object make it easier to subsequently reach towards another red object? In the current study, we asked observers to find the uniquely colored target object on each trial. Randomly intermixed pre-trial cues indicated the mode of action: either an eye movement or a visually guided reach movement to the target. In Experiment 1, we found that priming of popout, reflected in faster responses following repetition of the target color on consecutive trials, occurred regardless of whether the effector was repeated from the previous trial or not. In Experiment 2, we examined whether an inhibitory selection bias away from a feature could transfer across effectors. While priming of popout reflects both enhancement of the repeated target features and suppression of the repeated distractor features, the distractor previewing effect isolates a purely inhibitory component of target selection in which a previewed color is presented in a homogenous display and subsequently inhibited. Much like priming of popout, intertrial suppression biases in the distractor previewing effect transferred across effectors. Together, these results suggest that biases for target selection driven by recent trial history transfer across effectors. This indicates that representations in memory that bias attention towards or away from specific features are largely independent from their associated actions.

  6. Structural Analysis of Iac Repressor Bound to Allosteric Effectors

    SciTech Connect

    Daber,R.; Stayrook, S.; Rosenberg, A.; Lewis, M.

    2007-01-01

    The lac operon is a model system for understanding how effector molecules regulate transcription and are necessary for allosteric transitions. The crystal structures of the lac repressor bound to inducer and anti-inducer molecules provide a model for how these small molecules can modulate repressor function. The structures of the apo repressor and the repressor bound to effector molecules are compared in atomic detail. All effectors examined here bind to the repressor in the same location and are anchored to the repressor through hydrogen bonds to several hydroxyl groups of the sugar ring. Inducer molecules form a more extensive hydrogen-bonding network compared to anti-inducers and neutral effector molecules. The structures of these effector molecules suggest that the O6 hydroxyl on the galactoside is essential for establishing a water-mediated hydrogen bonding network that bridges the N-terminal and C-terminal sub-domains. The altered hydrogen bonding can account in part for the different structural conformations of the repressor, and is vital for the allosteric transition.

  7. Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases.

    PubMed

    Cai, Yujia; Bak, Rasmus O; Mikkelsen, Jacob Giehm

    2014-04-24

    Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in 'all-in-one' lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases.DOI: http://dx.doi.org/10.7554/eLife.01911.001.

  8. Effector-triggered defence against apoplastic fungal pathogens

    PubMed Central

    Stotz, Henrik U.; Mitrousia, Georgia K.; de Wit, Pierre J.G.M.; Fitt, Bruce D.L.

    2014-01-01

    R gene-mediated host resistance against apoplastic fungal pathogens is not adequately explained by the terms pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) or effector-triggered immunity (ETI). Therefore, it is proposed that this type of resistance is termed ‘effector-triggered defence’ (ETD). Unlike PTI and ETI, ETD is mediated by R genes encoding cell surface-localised receptor-like proteins (RLPs) that engage the receptor-like kinase SOBIR1. In contrast to this extracellular recognition, ETI is initiated by intracellular detection of pathogen effectors. ETI is usually associated with fast, hypersensitive host cell death, whereas ETD often triggers host cell death only after an elapsed period of endophytic pathogen growth. In this opinion, we focus on ETD responses against foliar fungal pathogens of crops. PMID:24856287

  9. Structural Insights into Rab27 Recruitment by its Effectors

    NASA Astrophysics Data System (ADS)

    M. G. Chavas, Leonard; Ihara, Kentaro; Kawasaki, Masato; Wakatsuki, Soichi

    An increasing number of Rab GTPases associated with partial dysfunction has been linked to several human diseases characterized by a diminution in vesicle transport. Due to its direct implication in human disorders, the Rab27 subfamily is considered as a standard for vesicle docking studies. By which mechanism Rab27 effectors distinguish among the pool of Rab GTPases? What is the underneath machinery rendering the interaction of eleven distinct effectors specific of Rab27 when compared to other Rabs of the secretory pathway? By solving the X-ray structures of Rab27, both in its inactive form and active form bound to the effector protein Slp2-a, attempts have been given to unravel the molecular basis of regulation of the delivering process of vesicles to fusion by the Rab27 subfamily.

  10. Signaling in Effector Lymphocytes: Insights toward Safer Immunotherapy

    PubMed Central

    Rajasekaran, Kamalakannan; Riese, Matthew J.; Rao, Sridhar; Wang, Li; Thakar, Monica S.; Sentman, Charles L.; Malarkannan, Subramaniam

    2016-01-01

    Receptors on T and NK cells systematically propagate highly complex signaling cascades that direct immune effector functions, leading to protective immunity. While extensive studies have delineated hundreds of signaling events that take place upon receptor engagement, the precise molecular mechanism that differentially regulates the induction or repression of a unique effector function is yet to be fully defined. Such knowledge can potentiate the tailoring of signal transductions and transform cancer immunotherapies. Targeted manipulations of signaling cascades can augment one effector function such as antitumor cytotoxicity while contain the overt generation of pro-inflammatory cytokines that contribute to treatment-related toxicity such as “cytokine storm” and “cytokine-release syndrome” or lead to autoimmune diseases. Here, we summarize how individual signaling molecules or nodes may be optimally targeted to permit selective ablation of toxic immune side effects. PMID:27242783

  11. Diacylglycerol Kinases in T Cell Tolerance and Effector Function

    PubMed Central

    Chen, Shelley S.; Hu, Zhiming; Zhong, Xiao-Ping

    2016-01-01

    Diacylglycerol kinases (DGKs) are a family of enzymes that regulate the relative levels of diacylglycerol (DAG) and phosphatidic acid (PA) in cells by phosphorylating DAG to produce PA. Both DAG and PA are important second messengers cascading T cell receptor (TCR) signal by recruiting multiple effector molecules, such as RasGRP1, PKCθ, and mTOR. Studies have revealed important physiological functions of DGKs in the regulation of receptor signaling and the development and activation of immune cells. In this review, we will focus on recent progresses in our understanding of two DGK isoforms, α and ζ, in CD8 T effector and memory cell differentiation, regulatory T cell development and function, and invariant NKT cell development and effector lineage differentiation. PMID:27891502

  12. Effector proteins that modulate plant--insect interactions.

    PubMed

    Hogenhout, Saskia A; Bos, Jorunn I B

    2011-08-01

    Insect herbivores have highly diverse life cycles and feeding behaviors. They establish close interactions with their plant hosts and suppress plant defenses. Chewing herbivores evoke characteristic defense responses distinguishable from general mechanical damage. In addition, piercing-sucking hemipteran insects display typical feeding behavior that suggests active suppression of plant defense responses. Effectors that modulate plant defenses have been identified in the saliva of these insects. Tools for high-throughput effector identification and functional characterization have been developed. In addition, in some insect species it is possible to silence gene expression by RNAi. Together, this technological progress has enabled the identification of insect herbivore effectors and their targets that will lead to the development of novel strategies for pest resistances in plants.

  13. Effector and suppressor T cells in celiac disease.

    PubMed

    Mazzarella, Giuseppe

    2015-06-28

    Celiac disease (CD) is a T-cell mediated immune disease in which gliadin-derived peptides activate lamina propria effector CD4+ T cells. This activation leads to the release of cytokines, compatible with a Th1-like pattern, which play a crucial role in the pathogenesis of CD, controlling many aspects of the inflammatory immune response. Recent studies have shown that a novel subset of effector T cells, characterized by expression of high levels of IL-17A, termed Th17 cells, plays a pathogenic role in CD. While these effector T cell subsets produce proinflammatory cytokines, which cause substantial tissue injury in vivo in CD, recent studies have suggested the existence of additional CD4(+) T cell subsets with suppressor functions. These subsets include type 1 regulatory T cells and CD25(+)CD4(+) regulatory T cells, expressing the master transcription factor Foxp3, which have important implications for disease progression.

  14. Yersinia type III effectors perturb host innate immune responses

    PubMed Central

    Pha, Khavong; Navarro, Lorena

    2016-01-01

    The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type III secretion system (T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp. (Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gram-negative bacteria that share in common a 70 kb virulence plasmid which encodes the T3SS. Translocation of the Yersinia effector proteins (YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia

  15. The genome editing revolution: A CRISPR-Cas TALE off-target story.

    PubMed

    Stella, Stefano; Montoya, Guillermo

    2016-07-01

    In the last 10 years, we have witnessed a blooming of targeted genome editing systems and applications. The area was revolutionized by the discovery and characterization of the transcription activator-like effector proteins, which are easier to engineer to target new DNA sequences than the previously available DNA binding templates, zinc fingers and meganucleases. Recently, the area experimented a quantum leap because of the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (clustered regularly interspaced short palindromic sequence). This ribonucleoprotein complex protects bacteria from invading DNAs, and it was adapted to be used in genome editing. The CRISPR ribonucleic acid (RNA) molecule guides to the specific DNA site the Cas9 nuclease to cleave the DNA target. Two years and more than 1000 publications later, the CRISPR-Cas system has become the main tool for genome editing in many laboratories. Currently the targeted genome editing technology has been used in many fields and may be a possible approach for human gene therapy. Furthermore, it can also be used to modifying the genomes of model organisms for studying human pathways or to improve key organisms for biotechnological applications, such as plants, livestock genome as well as yeasts and bacterial strains.

  16. Targeted Myostatin Gene Editing in Multiple Mammalian Species Directed by a Single Pair of TALE Nucleases.

    PubMed

    Xu, Li; Zhao, Piming; Mariano, Andrew; Han, Renzhi

    2013-07-30

    Myostatin (MSTN) is a negative regulator of skeletal muscle mass. Strategies to block myostatin signaling pathway have been extensively pursued to increase muscle mass in various disease settings including muscular dystrophy. Here, we report a new class of reagents based on transcription activator-like effector nucleases (TALENs) to disrupt myostatin expression at the genome level. We designed a pair of MSTN TALENs to target a highly conserved sequence in the coding region of the myostatin gene. We demonstrate that codelivery of these MSTN TALENs induce highly specific and efficient gene disruption in a variety of human, cattle, and mouse cells. Based upon sequence analysis, this pair of TALENs is expected to be functional in many other mammalian species. Moreover, we demonstrate that these MSTN TALENs can facilitate targeted integration of a mCherry expression cassette or a larger muscular dystrophy gene (dysferlin) expression cassette into the MSTN locus in mouse or human cells. Therefore, targeted editing of the myostatin gene using our highly specific and efficient TALEN pair would facilitate cell engineering, allowing potential use in translational research for cell-based therapy.Molecular Therapy-Nucleic Acids (2013) 2, e112; doi:10.1038/mtna.2013.39; published online 30 July 2013.

  17. Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases.

    PubMed

    Moon, JoonHo; Lee, Choongil; Kim, Su Jin; Choi, Ji-Yei; Lee, Byeong Chun; Kim, Jin-Soo; Jang, Goo

    2014-05-27

    Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (~24 hours) or their diameter (< 20 µm) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells.

  18. How Do Filamentous Pathogens Deliver Effector Proteins into Plant Cells?

    PubMed Central

    Petre, Benjamin; Kamoun, Sophien

    2014-01-01

    Fungal and oomycete plant parasites are among the most devastating pathogens of food crops. These microbes secrete effector proteins inside plant cells to manipulate host processes and facilitate colonization. How these effectors reach the host cytoplasm remains an unclear and debated area of plant research. In this article, we examine recent conflicting findings that have generated discussion in the field. We also highlight promising approaches based on studies of both parasite and host during infection. Ultimately, this knowledge may inform future broad spectrum strategies for protecting crops from such pathogens. PMID:24586116

  19. Nanorobotic end-effectors: Design, fabrication, and in situ characterization

    NASA Astrophysics Data System (ADS)

    Fan, Zheng

    Nano-robotic end-effectors have promising applications for nano-fabrication, nano-manufacturing, nano-optics, nano-medical, and nano-sensing; however, low performances of the conventional end-effectors have prevented the widespread utilization of them in various fields. There are two major difficulties in developing the end-effectors: their nano-fabrication and their advanced characterization in the nanoscale. Here we introduce six types of end-effectors: the nanotube fountain pen (NFP), the super-fine nanoprobe, the metal-filled carbon nanotube (m CNT)-based sphere-on-pillar (SOP) nanoantennas, the tunneling nanosensor, and the nanowire-based memristor. The investigations on the NFP are focused on nano-fluidics and nano-fabrications. The NFP could direct write metallic "inks" and fabricating complex metal nanostructures from 0D to 3D with a position servo control, which is critically important to future large-scale, high-throughput nanodevice production. With the help of NFP, we could fabricate the end-effectors such as super-fine nanoprobe and m CNT-based SOP nanoantennas. Those end-effectors are able to detect local flaws or characterize the electrical/mechanical properties of the nanostructure. Moreover, using electron-energy-loss-spectroscopy (EELS) technique during the operation of the SOP optical antenna opens a new basis for the application of nano-robotic end-effectors. The technique allows advanced characterization of the physical changes, such as carrier diffusion, that are directly responsible for the device's properties. As the device was coupled with characterization techniques of scanning-trasmission-electron-microscopy (STEM), the development of tunneling nanosensor advances this field of science into quantum world. Furthermore, the combined STEM-EELS technique plays an important role in our understanding of the memristive switching performance in the nanowire-based memristor. The developments of those nano-robotic end-effectors expend the study

  20. Visual End-Effector Position Error Compensation for Planetary Robotics

    NASA Technical Reports Server (NTRS)

    Bajracharya, Max; DiCicco, Matthew; Backes, Paul; Nickels, Kevin

    2007-01-01

    This paper describes a vision-guided manipulation algorithm that improves arm end-effector positioning to subpixel accuracy and meets the highly restrictive imaging and computational constraints of a planetary robotic flight system. Analytical, simulation-based, and experimental analyses of the algorithm's effectiveness and sensitivity to camera and arm model error is presented along with results on several prototype research systems and 'ground-in-the-loop' technology experiments on the Mars Exploration Rover (MER) vehicles. A computationally efficient and robust subpixel end-effector fiducial detector that is instrumental to the algorithm's ability to achieve high accuracy is also described along with its validation results on MER data.

  1. Hemipteran and dipteran pests: Effectors and plant host immune regulators.

    PubMed

    Kaloshian, Isgouhi; Walling, Linda L

    2016-04-01

    Hemipteran and dipteran insects have behavioral, cellular and chemical strategies for evading or coping with the host plant defenses making these insects particularly destructive pests worldwide. A critical component of a host plant's defense to herbivory is innate immunity. Here we review the status of our understanding of the receptors that contribute to perception of hemipteran and dipteran pests and highlight the gaps in our knowledge in these early events in immune signaling. We also highlight recent advances in identification of the effectors that activate pattern-triggered immunity and those involved in effector-triggered immunity.

  2. LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell

    PubMed Central

    Sperschneider, Jana; Catanzariti, Ann-Maree; DeBoer, Kathleen; Petre, Benjamin; Gardiner, Donald M.; Singh, Karam B.; Dodds, Peter N.; Taylor, Jennifer M.

    2017-01-01

    Pathogens secrete effector proteins and many operate inside plant cells to enable infection. Some effectors have been found to enter subcellular compartments by mimicking host targeting sequences. Although many computational methods exist to predict plant protein subcellular localization, they perform poorly for effectors. We introduce LOCALIZER for predicting plant and effector protein localization to chloroplasts, mitochondria, and nuclei. LOCALIZER shows greater prediction accuracy for chloroplast and mitochondrial targeting compared to other methods for 652 plant proteins. For 107 eukaryotic effectors, LOCALIZER outperforms other methods and predicts a previously unrecognized chloroplast transit peptide for the ToxA effector, which we show translocates into tobacco chloroplasts. Secretome-wide predictions and confocal microscopy reveal that rust fungi might have evolved multiple effectors that target chloroplasts or nuclei. LOCALIZER is the first method for predicting effector localisation in plants and is a valuable tool for prioritizing effector candidates for functional investigations. LOCALIZER is available at http://localizer.csiro.au/. PMID:28300209

  3. Steady-State and Kinetics-Based Affinity Determination in Effector-Effector Target Interactions.

    PubMed

    Reinhard, André; Nürnberger, Thorsten

    2017-01-01

    Dissecting the functional basis of pathogenicity and resistance in the context of plant innate immunity benefits greatly from detailed knowledge about biomolecular interactions, as both resistance and virulence depend on specific interactions between pathogen and host biomolecules. While in vivo systems provide biological context to host-pathogen interactions, these experiments typically cannot provide quantitative biochemical characterization of biomolecular interactions. However, in many cases, the biological function does not only depend on whether an interaction occurs at all, but rather on the "intensity" of the interaction, as quantified by affinity. Specifically, microbial effector proteins may bind more than one host target to exert virulence functions, and looking at these interactions more closely than would be possible in a purely black-and-white qualitative assay (as classically based on plant or yeast systems) can reveal new insights into the evolutionary arms race between host and pathogen. Recent advances in biomolecular interaction assays that can be performed in vitro allow quantification of binding events with ever greater fidelity and application range. Here, we describe assays based on microscale thermophoresis (MST) and surface plasmon resonance (SPR). Using these technologies allows affinity determination both in steady-state and in kinetic configurations, providing two conceptually independent pathways to arrive at quantitative affinity data describing the interactions of pathogen and host biomolecules.

  4. Robotic End Effectors for Hard-Rock Climbing

    NASA Technical Reports Server (NTRS)

    Kennedy, Brett; Leger, Patrick

    2004-01-01

    Special-purpose robot hands (end effectors) now under development are intended to enable robots to traverse cliffs much as human climbers do. Potential applications for robots having this capability include scientific exploration (both on Earth and other rocky bodies in space), military reconnaissance, and outdoor search and rescue operations. Until now, enabling robots to traverse cliffs has been considered too difficult a task because of the perceived need of prohibitively sophisticated planning algorithms as well as end effectors as dexterous as human hands. The present end effectors are being designed to enable robots to attach themselves to typical rock-face features with less planning and simpler end effectors. This advance is based on the emulation of the equipment used by human climbers rather than the emulation of the human hand. Climbing-aid equipment, specifically cams, aid hooks, and cam hooks, are used by sport climbers when a quick ascent of a cliff is desired (see Figure 1). Currently two different end-effector designs have been created. The first, denoted the simple hook emulator, consists of three "fingers" arranged around a central "palm." Each finger emulates the function of a particular type of climbing hook (aid hook, wide cam hook, and a narrow cam hook). These fingers are connected to the palm via a mechanical linkage actuated with a leadscrew/nut. This mechanism allows the fingers to be extended or retracted. The second design, denoted the advanced hook emulator (see Figure 2), shares these features, but it incorporates an aid hook and a cam hook into each finger. The spring-loading of the aid hook allows the passive selection of the type of hook used. The end effectors can be used in several different modes. In the aid-hook mode, the aid hook on one of the fingers locks onto a horizontal ledge while the other two fingers act to stabilize the end effector against the cliff face. In the cam-hook mode, the broad, flat tip of the cam hook is

  5. Hand to Mouth: Automatic Imitation across Effector Systems

    ERIC Educational Resources Information Center

    Leighton, Jane; Heyes, Cecilia

    2010-01-01

    The effector dependence of automatic imitation was investigated using a stimulus-response compatibility (SRC) procedure during which participants were required to make an open or closed response with their hand or their mouth. The correct response for each trial was indicated by a pair of letters in Experiments 1 and 2 and by a colored square in…

  6. Plasmodium cellular effector mechanisms and the hepatic microenvironment

    PubMed Central

    Frevert, Ute; Krzych, Urszula

    2015-01-01

    Plasmodium falciparum malaria remains one of the most serious health problems globally. Immunization with attenuated parasites elicits multiple cellular effector mechanisms capable of eliminating Plasmodium liver stages. However, malaria liver stage (LS) immunity is complex and the mechanisms effector T cells use to locate the few infected hepatocytes in the large liver in order to kill the intracellular LS parasites remain a mystery to date. Here, we review our current knowledge on the behavior of CD8 effector T cells in the hepatic microvasculature, in malaria and other hepatic infections. Taking into account the unique immunological and lymphogenic properties of the liver, we discuss whether classical granule-mediated cytotoxicity might eliminate infected hepatocytes via direct cell contact or whether cytokines might operate without cell–cell contact and kill Plasmodium LSs at a distance. A thorough understanding of the cellular effector mechanisms that lead to parasite death hence sterile protection is a prerequisite for the development of a successful malaria vaccine to protect the 40% of the world’s population currently at risk of Plasmodium infection. PMID:26074888

  7. Modulation of hemoglobin dynamics by an allosteric effector

    PubMed Central

    Maccarini, Marco; Fouquet, Peter; Ho, Nancy T.; Ho, Chien; Makowski, Lee

    2017-01-01

    Abstract Hemoglobin (Hb) is an extensively studied paradigm of proteins that alter their function in response to allosteric effectors. Models of its action have been used as prototypes for structure‐function relationships in many proteins, and models for the molecular basis of its function have been deeply studied and extensively argued. Recent reports suggest that dynamics may play an important role in its function. Relatively little is known about the slow, correlated motions of hemoglobin subunits in various structural states because experimental and computational strategies for their characterization are challenging. Allosteric effectors such as inositol hexaphosphate (IHP) bind to both deoxy‐Hb and HbCO, albeit at different sites, leading to a lowered oxygen affinity. The manner in which these effectors impact oxygen binding is unclear and may involve changes in structure, dynamics or both. Here we use neutron spin echo measurements accompanied by wide‐angle X‐ray scattering to show that binding of IHP to HbCO results in an increase in the rate of coordinated motions of Hb subunits relative to one another with little if any change in large scale structure. This increase of large‐scale dynamics seems to be coupled with a decrease in the average magnitude of higher frequency modes of individual residues. These observations indicate that enhanced dynamic motions contribute to the functional changes induced by IHP and suggest that they may be responsible for the lowered oxygen affinity triggered by these effectors. PMID:27977887

  8. Type IV secretion system of Brucella spp. and its effectors

    PubMed Central

    Ke, Yuehua; Wang, Yufei; Li, Wengfeng; Chen, Zeliang

    2015-01-01

    Brucella spp. are intracellular bacterial pathogens that cause infection in domestic and wild animals. They are often used as model organisms to study intracellular bacterial infections. Brucella VirB T4SS is a key virulence factor that plays important roles in mediating intracellular survival and manipulating host immune response to infection. In this review, we discuss the roles of Brucella VirB T4SS and 15 effectors that are proposed to be crucial for Brucella pathogenesis. VirB T4SS regulates the inflammation response and manipulates vesicle trafficking inside host cells. VirB T4SS also plays crucial roles in the inhibition of the host immune response and intracellular survival during infection. Here, we list the key molecular events in the intracellular life cycle of Brucella that are potentially targeted by the VirB T4SS effectors. Elucidating the functions of these effectors will help clarify the molecular role of T4SS during infection. Furthermore, studying the effectors secreted by Brucella spp. might provide insights into the mechanisms used by the bacteria to hijack the host signaling pathways and aid in the development of better vaccines and therapies against brucellosis. PMID:26528442

  9. Electroporation of Functional Bacterial Effectors into Mammalian Cells

    DOE PAGES

    Sontag, Ryan L.; Mihai, Cosmin; Orr, Galya; ...

    2015-01-19

    Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

  10. Dissociating Visual Attention and Effector Selection in Spatial Precuing Tasks

    ERIC Educational Resources Information Center

    Adam, Jos J.; Pratt, Jay

    2004-01-01

    In this study the authors tested and rejected a recent proposal that response precuing effects depend on the spatial extent of the precues rather than on the number of response choices. Moreover, the authors tested and supported the hypothesis that the number of effectors in the response set is an important determinant of spatial precuing effects.…

  11. Type IV secretion system of Brucella spp. and its effectors.

    PubMed

    Ke, Yuehua; Wang, Yufei; Li, Wengfeng; Chen, Zeliang

    2015-01-01

    Brucella spp. are intracellular bacterial pathogens that cause infection in domestic and wild animals. They are often used as model organisms to study intracellular bacterial infections. Brucella VirB T4SS is a key virulence factor that plays important roles in mediating intracellular survival and manipulating host immune response to infection. In this review, we discuss the roles of Brucella VirB T4SS and 15 effectors that are proposed to be crucial for Brucella pathogenesis. VirB T4SS regulates the inflammation response and manipulates vesicle trafficking inside host cells. VirB T4SS also plays crucial roles in the inhibition of the host immune response and intracellular survival during infection. Here, we list the key molecular events in the intracellular life cycle of Brucella that are potentially targeted by the VirB T4SS effectors. Elucidating the functions of these effectors will help clarify the molecular role of T4SS during infection. Furthermore, studying the effectors secreted by Brucella spp. might provide insights into the mechanisms used by the bacteria to hijack the host signaling pathways and aid in the development of better vaccines and therapies against brucellosis.

  12. Ehrlichia Moonlighting Effectors and Interkingdom Interactions with the Mononuclear Phagocyte

    PubMed Central

    Dunphy, Paige Selvy; Luo, Tian; McBride, Jere W.

    2013-01-01

    Ehrlichia chaffeensis is an obligately intracellular gram negative bacterium with a small genome that thrives in mammalian mononuclear phagoctyes by exploiting eukaryotic processes. Herein, we discuss the latest findings on moonlighting tandem repeat protein effectors and their secretion mechanisms, and novel molecular interkingdom interactions that provide insight into the intracellular pathobiology of ehrlichiae. PMID:24141087

  13. Structure Analysis Uncovers a Highly Diverse but Structurally Conserved Effector Family in Phytopathogenic Fungi

    PubMed Central

    Gracy, Jérome; Fournier, Elisabeth; Kroj, Thomas; Padilla, André

    2015-01-01

    Phytopathogenic ascomycete fungi possess huge effector repertoires that are dominated by hundreds of sequence-unrelated small secreted proteins. The molecular function of these effectors and the evolutionary mechanisms that generate this tremendous number of singleton genes are largely unknown. To get a deeper understanding of fungal effectors, we determined by NMR spectroscopy the 3-dimensional structures of the Magnaporthe oryzae effectors AVR1-CO39 and AVR-Pia. Despite a lack of sequence similarity, both proteins have very similar 6 β-sandwich structures that are stabilized in both cases by a disulfide bridge between 2 conserved cysteins located in similar positions of the proteins. Structural similarity searches revealed that AvrPiz-t, another effector from M. oryzae, and ToxB, an effector of the wheat tan spot pathogen Pyrenophora tritici-repentis have the same structures suggesting the existence of a family of sequence-unrelated but structurally conserved fungal effectors that we named MAX-effectors (Magnaporthe Avrs and ToxB like). Structure-informed pattern searches strengthened this hypothesis by identifying MAX-effector candidates in a broad range of ascomycete phytopathogens. Strong expansion of the MAX-effector family was detected in M. oryzae and M. grisea where they seem to be particularly important since they account for 5–10% of the effector repertoire and 50% of the cloned avirulence effectors. Expression analysis indicated that the majority of M. oryzae MAX-effectors are expressed specifically during early infection suggesting important functions during biotrophic host colonization. We hypothesize that the scenario observed for MAX-effectors can serve as a paradigm for ascomycete effector diversity and that the enormous number of sequence-unrelated ascomycete effectors may in fact belong to a restricted set of structurally conserved effector families. PMID:26506000

  14. Genome-Scale Identification of Legionella pneumophila Effectors Using a Machine Learning Approach

    PubMed Central

    Burstein, David; Zusman, Tal; Degtyar, Elena; Viner, Ram; Segal, Gil; Pupko, Tal

    2009-01-01

    A large number of highly pathogenic bacteria utilize secretion systems to translocate effector proteins into host cells. Using these effectors, the bacteria subvert host cell processes during infection. Legionella pneumophila translocates effectors via the Icm/Dot type-IV secretion system and to date, approximately 100 effectors have been identified by various experimental and computational techniques. Effector identification is a critical first step towards the understanding of the pathogenesis system in L. pneumophila as well as in other bacterial pathogens. Here, we formulate the task of effector identification as a classification problem: each L. pneumophila open reading frame (ORF) was classified as either effector or not. We computationally defined a set of features that best distinguish effectors from non-effectors. These features cover a wide range of characteristics including taxonomical dispersion, regulatory data, genomic organization, similarity to eukaryotic proteomes and more. Machine learning algorithms utilizing these features were then applied to classify all the ORFs within the L. pneumophila genome. Using this approach we were able to predict and experimentally validate 40 new effectors, reaching a success rate of above 90%. Increasing the number of validated effectors to around 140, we were able to gain novel insights into their characteristics. Effectors were found to have low G+C content, supporting the hypothesis that a large number of effectors originate via horizontal gene transfer, probably from their protozoan host. In addition, effectors were found to cluster in specific genomic regions. Finally, we were able to provide a novel description of the C-terminal translocation signal required for effector translocation by the Icm/Dot secretion system. To conclude, we have discovered 40 novel L. pneumophila effectors, predicted over a hundred additional highly probable effectors, and shown the applicability of machine learning algorithms for

  15. Active Flow Effectors for Noise and Separation Control

    NASA Technical Reports Server (NTRS)

    Turner, Travis L.

    2011-01-01

    New flow effector technology for separation control and enhanced mixing is based upon shape memory alloy hybrid composite (SMAHC) technology. The technology allows for variable shape control of aircraft structures through actively deformable surfaces. The flow effectors are made by embedding shape memory alloy actuator material in a composite structure. When thermally actuated, the flow effector def1ects into or out of the flow in a prescribed manner to enhance mixing or induce separation for a variety of applications, including aeroacoustic noise reduction, drag reduction, and f1ight control. The active flow effectors were developed for noise reduction as an alternative to fixed-configuration effectors, such as static chevrons, that cannot be optimized for airframe installation effects or variable operating conditions and cannot be retracted for off-design or fail-safe conditions. Benefits include: Increased vehicle control, overall efficiency, and reduced noise throughout all f1ight regimes, Reduced flow noise, Reduced drag, Simplicity of design and fabrication, Simplicity of control through direct current stimulation, autonomous re sponse to environmental heating, fast re sponse, and a high degree of geometric stability. The concept involves embedding prestrained SMA actuators on one side of the chevron neutral axis in order to generate a thermal moment and def1ect the structure out of plane when heated. The force developed in the host structure during def1ection and the aerodynamic load is used for returning the structure to the retracted position. The chevron design is highly scalable and versatile, and easily affords active and/or autonomous (environmental) control. The technology offers wide-ranging market applications, including aerospace, automotive, and any application that requires flow separation or noise control.

  16. Die another day: molecular mechanisms of effector-triggered immunity elicited by type III secreted effector proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial pathogens inject type III secreted effector (T3SE) proteins into their hosts where they display dual roles depending on the host genotype. T3SEs promote bacterial virulence in susceptible hosts, and elicit immunity in resistant hosts. T3SEs are typically recognized when they modify a host ...

  17. Opening the Ralstonia solanacearum type III effector tool box: insights into host cell subversion mechanisms.

    PubMed

    Deslandes, Laurent; Genin, Stephane

    2014-08-01

    Effectors delivered to host cells by the Type III secretion system are essential to Ralstonia solanacearum pathogenicity, as in several other plant pathogenic bacteria. The establishment of exhaustive effector repertoires in multiple R. solanacearum strains drew a first picture of the evolutionary dynamics of the pathogen effector suites. Effector repertoires are diversified, with a core of 20-30 effectors present in most of the strains and the obtention of mutants lacking one or more effector genes revealed the functional overlap among this effector network. Recent functional studies have provided insights into the ability of single effectors to manipulate the host proteasome, elicit cell death, trigger the expression of plant genes, and/or display biochemical activities on plant protein targets.

  18. Early effector cells survive the contraction phase in malaria infection and generate both central and effector memory T cells.

    PubMed

    Opata, Michael M; Carpio, Victor H; Ibitokou, Samad A; Dillon, Brian E; Obiero, Joshua M; Stephens, Robin

    2015-06-01

    CD4 T cells orchestrate immunity against blood-stage malaria. However, a major challenge in designing vaccines to the disease is poor understanding of the requirements for the generation of protective memory T cells (Tmem) from responding effector T cells (Teff) in chronic parasite infection. In this study, we use a transgenic mouse model with T cells specific for the merozoite surface protein (MSP)-1 of Plasmodium chabaudi to show that activated T cells generate three distinct Teff subsets with progressive activation phenotypes. The earliest observed Teff subsets (CD127(-)CD62L(hi)CD27(+)) are less divided than CD62L(lo) Teff and express memory genes. Intermediate (CD62L(lo)CD27(+)) effector subsets include the most multicytokine-producing T cells, whereas fully activated (CD62L(lo)CD27(-)) late effector cells have a terminal Teff phenotype (PD-1(+), Fas(hi), AnnexinV(+)). We show that although IL-2 promotes expansion, it actually slows terminal effector differentiation. Using adoptive transfer, we show that only early Teff survive the contraction phase and generate the terminal late Teff subsets, whereas in uninfected recipients, they become both central and effector Tmem. Furthermore, we show that progression toward full Teff activation is promoted by increased duration of infection, which in the long-term promotes Tem differentiation. Therefore, we have defined markers of progressive activation of CD4 Teff at the peak of malaria infection, including a subset that survives the contraction phase to make Tmem, and show that Ag and cytokine levels during CD4 T cell expansion influence the proportion of activated cells that can survive contraction and generate memory in malaria infection.

  19. End-Effector Development for the PIP Puck Handling Robot

    SciTech Connect

    Fowley, M.D.

    2001-01-31

    It has been decided that excess, weapons-grade plutonium shall be immobilized to prevent nuclear proliferation. The method of immobilization is to encapsulate the plutonium in a ceramic puck, roughly the size of a hockey puck, using a sintering process. This method has been officially identified as the Plutonium Immobilization Process (PIP). A Can-in-Canister storage method will be used to further immobilize the plutonium. The Can-in-Canister method uses the existing design of a Defense Waste Processing Facility (DWPF) canister to house the plutonium pucks. the process begins with several pucks being stacked in a stainless steel can. Several of the stainless steel cans are stacked in a cage-like magazine. Several of the magazines are then placed in a DWPF canister. The DWPF canister is then filled with molten glass containing high-level, radioactive waste from the DWPF vitrification process. The Can-in-Canister method makes reclamation of plutonium from the pucks technically difficult and highly undesirable. The mechanical requirements of the Can-in-Canister process, in conjunction with the amount of time required to immobilize the vast quantities of weapons-grade plutonium, will expose personnel to unnecessarily high levels of radiation if the processes were completed manually, in glove boxes. Therefore, automated equipment is designed into the process to reduce or eliminate personnel exposure. Robots are used whenever the automated handling operations become complicated. There are two such operations in the initial stages of the Can-in-Canister process, which required a six-axis robot. The first operation is a press unloading process. The second operation is a tray transfer process. To successfully accomplish the operational tasks described in the two operations, the end-effector of the robot must be versatile, lightweight, and rugged. As a result of these demands, an extensive development process was undertaken to design the optimum end-effector for these puck

  20. End-Effector Development for the PIP Puck Handling Robot

    SciTech Connect

    Fowley, M.D.

    2001-01-03

    It has been decided that excess, weapons-grade plutonium shall be immobilized to prevent nuclear proliferation. The method of immobilization is to encapsulate the plutonium in a ceramic puck, roughly the size of a hockey puck, using a sintering process. This method has been officially identified as the Plutonium Immobilization Process (PIP). A Can-in-Canister storage method will be used to further immobilize the plutonium. The Can-in-Canister method uses the existing design of a Defense Waste Processing Facility (DWPF) canister to house the plutonium pucks. the process begins with several pucks being stacked in a stainless steel can. Several of the stainless steel cans are stacked in a cage-like magazine. Several of the magazines are then placed in a DWPF canister. The DWPF canister is then filled with molten glass containing high-level, radioactive waste from the DWPF vitrification process. The Can-in-Canister method makes reclamation of plutonium from the pucks technically difficult and highly undesirable. The mechanical requirements of the Can-in-Canister process, in conjunction with the amount of time required to immobilize the vast quantities of weapons-grade plutonium, will expose personnel to unnecessarily high levels of radiation if the processes were completed manually, in glove boxes. Therefore, automated equipment is designed into the process to reduce or eliminate personnel exposure. Robots are used whenever the automated handling operations become complicated. There are two such operations in the initial stages of the Can-in-Canister process, which required a six-axis robot. The first operation is a press unloading process. The second operation is a tray transfer process. To successfully accomplish the operational tasks described in the two operations, the end-effector of the robot must be versatile, lightweight, and rugged. As a result of these demands, an extensive development process was undertaken to design the optimum end-effector for these puck

  1. Erwinia amylovora effector protein Eop1 suppresses PAMP-triggered immunity in Malus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Erwinia amylovora (Ea) utilizes a type three secretion system (T3SS) to deliver effector proteins into plant host cells. Several Ea effectors have been identified based on their sequence similarity to plant and animal bacterial pathogen effectors; however, the function of the majority of Ea effecto...

  2. Planning Keypress and Reaching Responses: Effects of Response Location and Number of Potential Effectors

    ERIC Educational Resources Information Center

    Adam, Jos J.; Taminiau, Bettine; van Veen, Natasja; Ament, Bart; Rijcken, Jons M.; Meijer, Kenneth; Pratt, Jay

    2008-01-01

    In previous work the authors argued that the potential number of effectors in the response set is crucial in discriminating (multiple-effector) keypress from (single-effector) reaching responses. It is not clear, however, what influence the locus of responding (on vs. off the stimulus location for reaching and keypressing, respectively) has on…

  3. Rapid and efficient genome-wide characterization of Xanthomonas TAL effector genes

    PubMed Central

    Yu, Yan-Hua; Lu, Ye; He, Yong-Qiang; Huang, Sheng; Tang, Ji-Liang

    2015-01-01

    Xanthomonas TALE transcriptional activators act as virulence or avirulence factors by activating host disease susceptibility or resistance genes. Their specificity is determined by a tandem repeat domain. Some Xanthomonas pathogens contain 10–30 TALEs per strain. Although TALEs play critical roles in pathogenesis, their studies have so far been limited to a few examples, due to their highly repetitive gene structure and extreme similarity among different members, which constrict sequencing and assembling. To facilitate TALE studies, we developed an efficient and rapid pipeline for genome-wide cloning of tal genes as many as possible from a strain. Here, we report the pipeline and its use to identify all 18 tal genes from a newly isolated strain of the rice pathogen Xathomonas oryzae. Target prediction revealed a number of potential rice targets including several notable genes such as genes encoding SWEET, WRKY, Hen1, and BAK1 proteins, which provide candidates for further experimental functional analysis of the TALEs. PMID:26271455

  4. Survey on Effects of Fairy Tales on Turkish Language Training from Secondary School Students' Perspective

    ERIC Educational Resources Information Center

    Kiliç, Yasin

    2015-01-01

    Fairy tale is one of the most important genres in literature which reflects childish sensitivity, feeds child's soul, enriches his/her imagination and prepares him/her for the future. Emerging as product of oral literature, fairy tales were used as an instrument of training in the past and they still have the same function today. Educators think…

  5. Forever Acting Alone: The Absence of Female Collaboration in "Grimms' Fairy Tales."

    ERIC Educational Resources Information Center

    Mendelson, Michael

    1997-01-01

    Explores the roles allocated to women in the famous collection. States that, although collaboration figures prominently in many of the more than 200 tales, the benefits of collective action are not extended to women. Examines the differences between male and female collaboration in the tales. Contemplates the implications of these differences for…

  6. The Ordinary and the Fabulous: An Introduction to Myths, Legends and Fairy Tales. Second Edition.

    ERIC Educational Resources Information Center

    Cook, Elizabeth

    Written for teachers, for students who intend to be teachers or librarians, and for storytellers in general, this book interprets the familiar legends and tales (Greek, Scandinavian, German, and Celtic myths and legends; Arthurian romances; the Old Testament; and fairy tales) and describes how they can best be told to children. Parallel accounts…

  7. Toward a new twist in Hox and TALE DNA-binding specificity.

    PubMed

    Merabet, Samir; Lohmann, Ingrid

    2015-02-09

    Hox proteins gain specificity by interacting with TALE-class cofactors. In a recent issue of Cell and in this issue of Developmental Cell, Crocker et al. (2015) and Amin et al. (2015), respectively, demonstrate that non-canonical Hox/TALE binding sequences play a major role in the regionalized regulation of target gene expression in vivo.

  8. Engaging with Mathematics in the Kindergarten. Orchestrating a Fairy Tale through Questioning and Use of Tools

    ERIC Educational Resources Information Center

    Carlsen, Martin

    2013-01-01

    The aim of this study is to analyse how a kindergarten teacher orchestrated a mathematical activity involving a fairy tale. Taking a sociocultural perspective on learning and development, naturally occurring talk-in-interaction has been analysed in order to scrutinise the subtleties of the orchestration. The fairy tale "Goldilocks and the…

  9. Oral Interpretation of C.S. Lewis'"Narnia Tales": A Refracting of "Pictures."

    ERIC Educational Resources Information Center

    Keefe, Carolyn

    "The Chronicles of Narnia" are a series of seven fairy tales written by C.S. Lewis that have become popular with both children and adults. Lewis points to five aspects of the fairy tale form that made the form suitable for expressing the images he saw. The aspects are: (1) no love interest; (2) no close psychology; (3) severe restraints…

  10. The Celebration of Death: Two Folk Tales about DEath. Mini-Module.

    ERIC Educational Resources Information Center

    African-American Inst., New York, NY. School Services Div.

    This module contains two African folk tales about death, two descriptions of African funerals, a lesson plan with 11 questions exploring the finality of and customs surrounding death, and a bibliography of five books which deal with African religious beliefs. The folk tales present concepts of death and immortality of the soul. The descriptions of…

  11. How Can This Be Cinderella if There Is No Glass Slipper? Native American "Fairy Tales."

    ERIC Educational Resources Information Center

    Stewart, Michelle Pagni

    2000-01-01

    Analysis of three Native American Cinderella-type tales finds that although each has flaws in depicting Native American culture, overall they are culturally accurate and respectful. Such tales can be used as teaching tools to help children understand Native American cultures and beliefs while making them aware of how culture and beliefs can easily…

  12. Individuals or Types: A Character Analysis of Hans Christian Andersen's Fairy Tales.

    ERIC Educational Resources Information Center

    Clement, Sherrie

    In a literary analysis of selected Hans Christian Andersen fairy tales, the characters were divided into three main types: human beings; plants, animals, and inanimate objects; and imaginary creations. A comparison was then made between Andersen's characters and those in traditional fairy tales. The findings revealed that Andersen succeeded in his…

  13. Understanding "A Tale of Two Cities": A Student Casebook to Issues, Sources, and Historical Documents.

    ERIC Educational Resources Information Center

    Newlin, George

    Charles Dickens' novel, "A Tale of Two Cities," does not waste a word in telling a touching, suspenseful tale set against the background of one of the bloodiest events in history, the French Revolution. This casebook's collection of historical documents, collateral readings, and commentary will promote interdisciplinary study of the…

  14. The Therapeutic Use of Fairy Tales with Adults in Group Therapy

    ERIC Educational Resources Information Center

    Brown, Nina W.

    2007-01-01

    Fairy tales are not just for children. They can hold important messages for people of all ages, as well as for those with special needs. For example, people who present for therapy reporting such issues as fear of abandonment, sibling rivalry, self-esteem, and lack of meaning in life, among others, can benefit from using fairy tales as a…

  15. If the Shoe Fits: Virtue and Absolute Beauty in Fairy Tale Drama.

    ERIC Educational Resources Information Center

    Thompson, Melissa C.

    2000-01-01

    Examines five versions of the Cinderella story. Argues that children's fairy tale drama presents female beauty as a moral absolute, not merely as a physical characteristic. Cites feminist theories of the female body to demonstrate ways in which fairy tale drama establishes female beauty as an unchanging, "universal" concept. Considers…

  16. Interferon-inducible effector mechanisms in cell-autonomous immunity.

    PubMed

    MacMicking, John D

    2012-04-25

    Interferons (IFNs) induce the expression of hundreds of genes as part of an elaborate antimicrobial programme designed to combat infection in all nucleated cells - a process termed cell-autonomous immunity. As described in this Review, recent genomic and subgenomic analyses have begun to assign functional properties to novel IFN-inducible effector proteins that restrict bacteria, protozoa and viruses in different subcellular compartments and at different stages of the pathogen life cycle. Several newly described host defence factors also participate in canonical oxidative and autophagic pathways by spatially coordinating their activities to enhance microbial killing. Together, these IFN-induced effector networks help to confer vertebrate host resistance to a vast and complex microbial world.

  17. Interchangeable end effector tools utilized on the protoflight manipulator arm

    NASA Technical Reports Server (NTRS)

    1987-01-01

    A subset of teleoperator and effector tools was designed, fabricated, delivered and successfully demonstrated on the Marshall Space Flight Center (MSFC) protoflight manipulator arm (PFMA). The tools delivered included a rotary power tool with interchangeable collets and two fluid coupling mate/demate tools; one for a Fairchild coupling and the other for a Purolator coupling. An electrical interface connector was also provided for the rotary power tool. A tool set, from which the subset was selected, for performing on-orbit satellite maintenance was identified and conceptionally designed. Maintenance requirements were synthesized, evaluated and prioritized to develop design requirements for a set of end effector tools representative of those needed to provide on-orbit maintenance of satellites to be flown in the 1986 to 2000 timeframe.

  18. A robot end effector exchange mechanism for space applications

    NASA Technical Reports Server (NTRS)

    Gorin, Barney F.

    1990-01-01

    Efficient robot operation requires the use of specialized end effectors or tools for tasks. In spacecraft applications, the microgravity environment precludes the use of gravitational forces to retain the tools in holding fixture. As a result of this, a retention mechanism which forms a part of the tool storage container is required. A unique approach to this problem has resulted in the development of an end effector exchange mechanism that meets the requirements for spaceflight applications while avoiding the complexity usually involved. This mechanism uses multiple latching cams both on the manipulator and in the tool storage container, combined with a system of catch rings to provide retention in both locations and the required failure tolerance. Because of the cam configuration the mechanism operates passively, requiring no electrical commands except those needed to move the manipulator into position. Similarly, it inherently provides interlocks to prevent the release of one cam before its opposite number is engaged.

  19. TLR-exosomes exhibit distinct kinetics and effector function

    PubMed Central

    Srinivasan, Swetha; Su, Michelle; Ravishankar, Shashidhar; Moore, James; Head, PamelaSara; Dixon, J. Brandon; Vannberg, Fredrik

    2017-01-01

    The innate immune system is vital to rapidly responding to pathogens and Toll-like receptors (TLRs) are a critical component of this response. Nanovesicular exosomes play a role in immunity, but to date their exact contribution to the dissemination of the TLR response is unknown. Here we show that exosomes from TLR stimulated cells can largely recapitulate TLR activation in distal cells in vitro. We can abrogate the action-at-a-distance signaling of exosomes by UV irradiation, demonstrating that RNA is crucial for their effector function. We are the first to show that exosomes derived from poly(I:C) stimulated cells induce in vivo macrophage M1-like polarization within murine lymph nodes. These poly(I:C) exosomes demonstrate enhanced trafficking to the node and preferentially recruit neutrophils as compared to control exosomes. This work definitively establishes the differential effector function for exosomes in communicating the TLR activation state of the cell of origin. PMID:28290538

  20. Design and Implementation of Multifunctional Automatic Drilling End Effector

    NASA Astrophysics Data System (ADS)

    Wang, Zhanxi; Qin, Xiansheng; Bai, Jing; Tan, Xiaoqun; Li, Jing

    2017-03-01

    In order to realize the automatic drilling in aircraft assembly, a drilling end effector is designed by integrating the pressure unit, drilling unit, measurement unit, control system and frame structure. In order to reduce the hole deviation, this paper proposes a vertical normal adjustment program based on 4 laser distance sensors. The actual normal direction of workpiece surface can be calculated through the sensors measurements, and then robot posture is adjusted to realize the hole deviation correction. A base detection method is proposed to detect and locate the hole automatically by using the camera and the reference hole. The experiment results show that the position accuracy of the system is less than 0.3mm, and the normal precision is less than 0.5°. The drilling end effector and robot can greatly improve the efficiency of the aircraft parts and assembly quality, and reduce the product development cycle.

  1. Helicases in antiviral immunity: dual properties as sensors and effectors

    PubMed Central

    Ahmad, Sadeem; Hur, Sun

    2015-01-01

    Many helicases have a unique ability to couple cognate RNA binding to ATP hydrolysis, which can induce a large conformational change that affects its interaction with RNA, position along RNA, or oligomeric state. A growing number of these helicases contribute to the innate immune system, either as sensors that detect foreign nucleic acids and/or as effectors that directly participate in the clearance of such foreign species. We here discuss a few examples, including retinoic acid-inducible gene-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and Dicer, focusing on their dual functions as both sensors and effectors. We will also discuss the closely related, but less understood helicases, laboratory of genetics and physiology 2 (LGP2) and Dicer-related helicase-1 and -3 (DRH-1 and -3). PMID:26410598

  2. Identification of novel type III effectors using latent Dirichlet allocation.

    PubMed

    Yang, Yang

    2012-01-01

    Among the six secretion systems identified in Gram-negative bacteria, the type III secretion system (T3SS) plays important roles in the disease development of pathogens. T3SS has attracted a great deal of research interests. However, the secretion mechanism has not been fully understood yet. Especially, the identification of effectors (secreted proteins) is an important and challenging task. This paper adopts machine learning methods to identify type III secreted effectors (T3SEs). We extract features from amino acid sequences and conduct feature reduction based on latent semantic information by using latent Dirichlet allocation model. The experimental results on Pseudomonas syringae data set demonstrate the good performance of the new methods.

  3. Review: Tales from Both Sides of the Brain.

    PubMed

    Landis, Theodor

    2015-01-01

    Our brain has two hemispheres that specialize in different jobs-the right side processes spatial and temporal information, and the left side controls speech and language. How these two sides come together to create one mind is explained by pioneering neuroscientist Michael Gazzaniga in his new book, Tales from Both Sides of the Brain : A Life in Neuroscience (Ecco/Harper Collins, 2015). Gazzaniga is director of the SAGE Center for the Study of the Mind at the University of California, Santa Barbara, and a Dana Alliance member.

  4. Review: Tales from Both Sides of the Brain

    PubMed Central

    Landis, Theodor

    2015-01-01

    Our brain has two hemispheres that specialize in different jobs—the right side processes spatial and temporal information, and the left side controls speech and language. How these two sides come together to create one mind is explained by pioneering neuroscientist Michael Gazzaniga in his new book, Tales from Both Sides of the Brain : A Life in Neuroscience (Ecco/Harper Collins, 2015). Gazzaniga is director of the SAGE Center for the Study of the Mind at the University of California, Santa Barbara, and a Dana Alliance member. PMID:27408671

  5. Xanthomonas and the TAL Effectors: Nature's Molecular Biologist.

    PubMed

    White, Frank

    2016-01-01

    Agrobacterium, due to the transfer of T-DNA to the host genome, is known as nature's genetic engineer. Once again, bacteria have led the way to newfound riches in biotechnology. Xanthomonas has emerged as nature's molecular biologist as the functional domains of the sequence-specific DNA transcription factors known as TAL effectors were characterized and associated with the cognate disease susceptibility and resistance genes of plants.

  6. A secreted fungal effector of Glomus intraradices promotes symbiotic biotrophy.

    PubMed

    Kloppholz, Silke; Kuhn, Hannah; Requena, Natalia

    2011-07-26

    Biotrophic fungi interacting with plants establish long-term relationships with their hosts to fulfill their life cycles. In contrast to necrotrophs, they need to contend with the defense mechanisms of the plant to develop within the host and feed on living cells. It is generally accepted that microbial pathogens produce and deliver a myriad of effector proteins to hijack the cellular program of their hosts. Arbuscular mycorrhizal (AM) fungi are the most widespread biotrophs of plant roots. We investigated whether AM fungi use effector proteins to short-circuit the plant defense program. Here we show that Glomus intraradices secretes a protein, SP7, that interacts with the pathogenesis-related transcription factor ERF19 in the plant nucleus. ERF19 is highly induced in roots by the fungal pathogen Colletotrichum trifolii as well as by several fungal extracts, but only transiently during mycorrhiza colonization. When constitutively expressed in roots, SP7 leads to higher mycorrhization while reducing the levels of C. trifolii-mediated defense responses. Furthermore, expression of SP7 in the rice blast fungus Magnaporthe oryzae attenuates root decay symptoms. Taken together, these results suggest that SP7 is an effector that contributes to develop the biotrophic status of AM fungi in roots by counteracting the plant immune program.

  7. Compact effector optics for processing in limited physical access situations.

    PubMed

    Kuhn, Andreas; Fox, Mahlen D T; French, Paul W; Hettrick, Simon; Hand, Duncan P; Shi, Yi-Wei; Matsuura, Yuji; Miyagi, Mitsunobu; Watkins, Kenneth G; Ireland, Clive L M; Jones, Julian D C

    2003-09-01

    A major advantage of fiber-optic beam delivery in laser materials processing is the ability to guide the laser power to the location where it is needed, leaving the laser itself remote and protected from the process. This is of special importance if the processing is to be performed in a hazardous environment. Particular problems are faced by the nuclear industry where weld repair and surface treatment work are required inside radioactive installations. By use of fiber beam delivery, only part of the delivery system and effector optics become contaminated, but the expensive laser system does not. However, in many cases the region where repair is required is not only radioactive but has only limited physical access, e.g., inside tubes or into corners, which prevents use of standard effector optics. We present a new design to deal with such constraints of a 2-mm outer diameter employing a hollow waveguide and gas shielding. This design is optically characterized and its performance assessed in welding and surface treatment applications. The potential of this compact effector optics in limited physical access situations is clearly demonstrated.

  8. AraC regulatory protein mutants with altered effector specificity.

    PubMed

    Tang, Shuang-Yan; Fazelinia, Hossein; Cirino, Patrick C

    2008-04-16

    The AraC regulatory protein of the Escherichia coli ara operon has been engineered to activate transcription in response to D-arabinose and not in response to its native effector L-arabinose. Two different AraC mutant libraries, each with four randomized binding pocket residues, were subjected to FACS-mediated dual screening using a GFP reporter. Both libraries yielded mutants with the desired switch in effector specificity, and one mutant we describe maintains tight repression in the absence of effector. The presence of 100 mM L-arabinose does not influence the response of the reported mutants to D-arabinose, and the mutants are not induced by other sugars tested (D-xylose, D-fucose, D-lyxose). Co-expression of the FucP transporter in E. coli enabled induction by D-arabinose in the 0.1 mM range. Our results demonstrate the power of dual screening for altering AraC inducer specificity and represent steps toward the design of customized in vivo molecular reporters and gene switches for metabolic engineering.

  9. A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin

    PubMed Central

    Liu, Yao; Zhu, Wenhan; Tan, Yunhao; Nakayasu, Ernesto S.; Staiger, Christopher J.

    2017-01-01

    Legionella pneumophila, the etiological agent of Legionnaires’ disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H95EXXH99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen. PMID:28129393

  10. Multiple recognition of RXLR effectors is associated with nonhost resistance of pepper against Phytophthora infestans

    PubMed Central

    Lee, Hyun-Ah; Kim, Shin-Young; Oh, Sang-Keun; Yeom, Seon-In; Kim, Saet-Byul; Kim, Myung-Shin; Kamoun, Sophien; Choi, Doil

    2014-01-01

    Nonhost resistance (NHR) is a plant immune response to resist most pathogens. The molecular basis of NHR is poorly understood, but recognition of pathogen effectors by immune receptors, a response known as effector-triggered immunity, has been proposed as a component of NHR. We performed transient expression of 54 Phytophthora infestansRXLR effectors in pepper (Capsicum annuum) accessions. We used optimized heterologous expression methods and analyzed the inheritance of effector-induced cell death in an F2 population derived from a cross between two pepper accessions. Pepper showed a localized cell death response upon inoculation with P. infestans, suggesting that recognition of effectors may contribute to NHR in this system. Pepper accessions recognized as many as 36 effectors. Among the effectors, PexRD8 and Avrblb2 induced cell death in a broad range of pepper accessions. Segregation of effector-induced cell death in an F2 population derived from a cross between two pepper accessions fit 15 : 1, 9 : 7 or 3 : 1 ratios, depending on the effector. Our genetic data suggest that a single or two independent/complementary dominant genes are involved in the recognition of RXLR effectors. Multiple loci recognizing a series of effectors may underpin NHR of pepper to P. infestans and confer resistance durability. PMID:24889686

  11. Multiple recognition of RXLR effectors is associated with nonhost resistance of pepper against Phytophthora infestans.

    PubMed

    Lee, Hyun-Ah; Kim, Shin-Young; Oh, Sang-Keun; Yeom, Seon-In; Kim, Saet-Byul; Kim, Myung-Shin; Kamoun, Sophien; Choi, Doil

    2014-08-01

    Nonhost resistance (NHR) is a plant immune response to resist most pathogens. The molecular basis of NHR is poorly understood, but recognition of pathogen effectors by immune receptors, a response known as effector-triggered immunity, has been proposed as a component of NHR. We performed transient expression of 54 Phytophthora infestansRXLR effectors in pepper (Capsicum annuum) accessions. We used optimized heterologous expression methods and analyzed the inheritance of effector-induced cell death in an F2 population derived from a cross between two pepper accessions. Pepper showed a localized cell death response upon inoculation with P. infestans, suggesting that recognition of effectors may contribute to NHR in this system. Pepper accessions recognized as many as 36 effectors. Among the effectors, PexRD8 and Avrblb2 induced cell death in a broad range of pepper accessions. Segregation of effector-induced cell death in an F2 population derived from a cross between two pepper accessions fit 15:1, 9:7 or 3:1 ratios, depending on the effector. Our genetic data suggest that a single or two independent/complementary dominant genes are involved in the recognition of RXLR effectors. Multiple loci recognizing a series of effectors may underpin NHR of pepper to P. infestans and confer resistance durability.

  12. Behind the lines–actions of bacterial type III effector proteins in plant cells

    PubMed Central

    Büttner, Daniela

    2016-01-01

    Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:28201715

  13. Plant-Pathogen Effectors: Cellular Probes Interfering with Plant Defenses in Spatial and Temporal Manners

    PubMed Central

    Toruño, Tania Y.; Stergiopoulos, Ioannis; Coaker, Gitta

    2017-01-01

    Plants possess large arsenals of immune receptors capable of recognizing all pathogen classes. To cause disease, pathogenic organisms must be able to overcome physical barriers, suppress or evade immune perception, and derive nutrients from host tissues. Consequently, to facilitate some of these processes, pathogens secrete effector proteins that promote colonization. This review covers recent advances in the field of effector biology, focusing on conserved cellular processes targeted by effectors from diverse pathogens. The ability of effectors to facilitate pathogen entry into the host interior, suppress plant immune perception, and alter host physiology for pathogen benefit is discussed. Pathogens also deploy effectors in a spatial and temporal manner, depending on infection stage. Recent advances have also enhanced our understanding of effectors acting in specific plant organs and tissues. Effectors are excellent cellular probes that facilitate insight into biological processes as well as key points of vulnerability in plant immune signaling networks. PMID:27359369

  14. Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 type III secretion effector genes reveal functional overlap among effectors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many bacterial pathogens of plants and animals disarm and remodel host cells by injecting large repertoires of effectors via the type III secretion system (T3SS). The repertoires of individual strains appear to function as robust systems that can tolerate loss of individual effectors with little or ...

  15. What makes folk tales unique: content familiarity, causal structure, scripts, or superstructures?

    PubMed

    McDaniel, M A; Hines, R J; Waddill, P J; Einstein, G O

    1994-01-01

    Requiring readers to re-order randomly ordered sentences into a coherent text significantly enhances recall relative to that in a read-only control condition for non-folk-tale texts but not for folk tales (Einstein, McDaniel, Owen, & Coté, 1990). Experiments 1-3 showed that embedding components of folk tales (e.g., causal structure, conventional scripts, content related to background knowledge) in non-folk-tale texts did not render sentence unscrambling ineffective for increasing recall. In Experiments 4a-4c, a folk tale was presented either as a fairy tale or as part of a newspaper article. Significant sentence unscrambling effects (in free recall) were not obtained in either presentation format, which implies that a story superstructure (a story grammar) does not contribute to the absence of the sentence unscrambling effect. It is suggested that understanding why the sentence unscrambling effect is absent for folk tales may require considering the functional role that narrative plays in socioculturally situated cognition.

  16. Identification of novel type III secretion effectors in Xanthomonas oryzae pv. oryzae.

    PubMed

    Furutani, Ayako; Takaoka, Minako; Sanada, Harumi; Noguchi, Yukari; Oku, Takashi; Tsuno, Kazunori; Ochiai, Hirokazu; Tsuge, Seiji

    2009-01-01

    Many gram-negative bacteria secrete so-called effector proteins via a type III secretion (T3S) system. Through genome screening for genes encoding potential T3S effectors, 60 candidates were selected from rice pathogen Xanthomonas oryzae pv. oryzae MAFF311018 using these criteria: i) homologs of known T3S effectors in plant-pathogenic bacteria, ii) genes with expression regulated by hrp regulatory protein HrpX, or iii) proteins with N-terminal amino acid patterns associated with T3S substrates of Pseudomonas syringae. Of effector candidates tested with the Bordetella pertussis calmodulin-dependent adenylate cyclase reporter for translocation into plant cells, 16 proteins were translocated in a T3S system-dependent manner. Of these 16 proteins, nine were homologs of known effectors in other plant-pathogenic bacteria and seven were not. Most of the effectors were widely conserved in Xanthomonas spp.; however, some were specific to X. oryzae. Interestingly, all these effectors were expressed in an HrpX-dependent manner, suggesting coregulation of effectors and the T3S system. In X. campestris pv. vesicatoria, HpaB and HpaC (HpaP in X. oryzae pv. oryzae) have a central role in recruiting T3S substrates to the secretion apparatus. Secretion of all but one effector was reduced in both HpaB() and HpaP() mutant strains, indicating that HpaB and HpaP are widely involved in efficient secretion of the effectors.

  17. Identification and Characterisation CRN Effectors in Phytophthora capsici Shows Modularity and Functional Diversity

    PubMed Central

    Stam, Remco; Jupe, Julietta; Howden, Andrew J. M.; Morris, Jenny A.; Boevink, Petra C.; Hedley, Pete E.; Huitema, Edgar

    2013-01-01

    Phytophthora species secrete a large array of effectors during infection of their host plants. The Crinkler (CRN) gene family encodes a ubiquitous but understudied class of effectors with possible but as of yet unknown roles in infection. To appreciate CRN effector function in Phytophthora, we devised a simple Crn gene identification and annotation pipeline to improve effector prediction rates. We predicted 84 full-length CRN coding genes and assessed CRN effector domain diversity in sequenced Oomycete genomes. These analyses revealed evidence of CRN domain innovation in Phytophthora and expansion in the Peronosporales. We performed gene expression analyses to validate and define two classes of CRN effectors, each possibly contributing to infection at different stages. CRN localisation studies revealed that P. capsici CRN effector domains target the nucleus and accumulate in specific sub-nuclear compartments. Phenotypic analyses showed that few CRN domains induce necrosis when expressed in planta and that one cell death inducing effector, enhances P. capsici virulence on Nicotiana benthamiana. These results suggest that the CRN protein family form an important class of intracellular effectors that target the host nucleus during infection. These results combined with domain expansion in hemi-biotrophic and necrotrophic pathogens, suggests specific contributions to pathogen lifestyles. This work will bolster CRN identification efforts in other sequenced oomycete species and set the stage for future functional studies towards understanding CRN effector functions. PMID:23536880

  18. p53 as an Effector or Inhibitor of Therapy Response.

    PubMed

    Ablain, Julien; Poirot, Brigitte; Esnault, Cécile; Lehmann-Che, Jacqueline; de Thé, Hugues

    2015-12-04

    Although integrity of the p53 signaling pathway in a given tumor was expected to be a critical determinant of response to therapies, most clinical studies failed to link p53 status and treatment outcome. Here, we present two opposite situations: one in which p53 is an essential effector of cure by targeted leukemia therapies and another one in advanced breast cancers in which p53 inactivation is required for the clinical efficacy of dose-dense chemotherapy. If p53 promotes or blocks therapy response, therapies must be tailored on its status in individual tumors.

  19. Telepresence Master Glove Controller For Dexterous Robotic End-Effectors

    NASA Astrophysics Data System (ADS)

    Fowler, A. M.; Joyce, R. R.; Britt, J. P.

    1987-03-01

    This paper describes recent research in the Aerospace Human Factors Research Division at NASA's Ames Research Center to develop a glove-like, control and data-recording device (DataGlove) that records and transmits to a host computerin real time, and at appropriate resolution, a numeric data-record of a user's hand/finger shape and dynamics. System configuration and performance specifications are detailed, and current research is discussed investigating its applications in operator control of dexterous robotic end-effectors and for use as a human factors research tool in evaluation of operator hand function requirements and performance in other specialized task environments.

  20. Telepresence master glove controller for dexterous robotic end-effectors

    NASA Technical Reports Server (NTRS)

    Fisher, Scott S.

    1987-01-01

    This paper describes recent research in the Aerospace Human Factors Research Division at NASA's Ames Research Center to develop a glove-like, control and data-recording device (DataGlove) that records and transmits to a host computer in real time, and at appropriate resolution, a numeric data-record of a user's hand/finger shape and dynamics. System configuration and performance specifications are detailed, and current research is discussed investigating its applications in operator control of dexterous robotic end-effectors and for use as a human factors research tool in evaluation of operator hand function requirements and performance in other specialized task environments.

  1. Exact positioning of the robotic arm end effector

    NASA Astrophysics Data System (ADS)

    Korepanov, Valery; Dudkin, Fedir

    2016-07-01

    Orbital service becomes a new challenge of space exploration. The necessity to introduce it is connected first of all with an attractive opportunity to prolong the exploitation terms of expensive commercial satellites by, e.g., refilling of fuel or changing batteries. Other application area is a fight with permanently increasing amount of space litter - defunct satellites, burnt-out rocket stages, discarded trash and other debris. Now more than few tens of thousands orbiting objects larger than 5-10 cm (or about 1 million junks larger than 1 cm) are a huge problem for crucial and costly satellites and manned vehicles. For example, in 2014 the International Space Station had to change three times its orbit to avoid collision with space debris. So the development of the concepts and actions related to removal of space debris or non-operational satellites with use of robotic arm of a servicing satellite is very actual. Such a technology is also applicable for unmanned exploratory missions in solar system, for example for collecting a variety of samples from a celestial body surface. Naturally, the robotic arm movements should be controlled with great accuracy at influence of its non-rigidity, thermal and other factors. In these circumstances often the position of the arm end effector has to be controlled with high accuracy. The possibility of coordinate determination for the robotic arm end effector with use of a low frequency active electromagnetic system has been considered in the presented report. The proposed design of such a system consists of a small magnetic dipole source, which is mounted inside of the arm end effector and two or three 3-component magnetic field sensors mounted on a servicing satellite body. The data from this set of 3-component magnetic field sensors, which are fixed relatively to the satellite body, allows use of the mathematical approach for determination of position and orientation of the magnetic dipole source. The theoretical

  2. Complement--tapping into new sites and effector systems.

    PubMed

    Kolev, Martin; Le Friec, Gaelle; Kemper, Claudia

    2014-12-01

    Complement is traditionally known to be a system of serum proteins that provide protection against pathogens through direct cell lysis and the mobilization of innate and adaptive immunity. However, recent work indicates that the complement system has additional physiological roles beyond those in host defence. In this Opinion article, we describe the new modes and locations of complement activation that enable it to interact with other cell effector systems, such as growth factor receptors, inflammasomes and metabolic pathways. We propose that the location of complement activation dictates its function.

  3. Tool use and the distalization of the end-effector

    PubMed Central

    Bonaiuto, James B.; Jacobs, Stéphane; Frey, Scott H.

    2009-01-01

    We review recent neurophysiological data from macaques and humans suggesting that the use of tools extends the internal representation of the actor’s hand, and relate it to our modeling of the visual control of grasping. We introduce the idea that, in addition to extending the body schema to incorporate the tool, tool use involves distalization of the end-effector from hand to tool. Different tools extend the body schema in different ways, with a displaced visual target and a novel, task-specific processing of haptic feedback to the hand. This distalization is critical in order to exploit the unique functional capacities engendered by complex tools. PMID:19347356

  4. Hacker within! Ehrlichia chaffeensis Effector Driven Phagocyte Reprogramming Strategy

    PubMed Central

    Lina, Taslima T.; Farris, Tierra; Luo, Tian; Mitra, Shubhajit; Zhu, Bing; McBride, Jere W.

    2016-01-01

    Ehrlichia chaffeensis is a small, gram negative, obligately intracellular bacterium that preferentially infects mononuclear phagocytes. It is the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection, and avoids host defenses are not well understood, but involve functionally relevant host-pathogen interactions associated with tandem and ankyrin repeat effector proteins. In this review, we discuss the recent advances in our understanding of the molecular and cellular mechanisms that underlie Ehrlichia host cellular reprogramming strategies that enable intracellular survival. PMID:27303657

  5. Duss Fairy Tales: some data from a new evaluation form.

    PubMed

    Mazzeschi, C; Lis, A; Calvo, V; Vallone, V; Superchi, E

    2001-12-01

    The purpose of this paper is to extend the research on the Duss Fairy Tales in an Italian sample. Attention has been paid, in particular, to the study of some variables identified in a newly devised schedule. The protocols were scored for four indexes: (1) the main hero of the stories, (2) number and types of characters, (3) number of emotions expressed, and (4) number of heroes' and characters' actions and behaviors. Subjects were 70 children aged 3.5 to 10.5 yr. enrolled in kindergartens and elementary schools in Italy. The relationships of scores with age and sex were also investigated. There was an increase across three age groups in the richness of stories in terms of emotions and characters' identification.

  6. CYPRUS TURKISH FAIRY TALES: GLIMPSE OF A HARMONIOUS PAST.

    PubMed

    Beyoğlu, Erdem

    2015-12-01

    On the island of Cyprus, believed to be the birthplace of Aphrodite, the Muslim minority (settled there following the Ottoman conquest of the island in 1571) and Orthodox Christians (the native majority) lived together in peace for hundreds of years. However, as a result of ethnic conflict in the late 1950s, the Muslim Cypriot Turks established their own political state in the north of the island in 1974, and Cyprus was divided into northern Turkish and southern Greek sections. This paper attempts to examine historical, religious, cultural and psychological aspects of the relationship between these two large groups, prior to recent conflicts, by studying fairy tales told by Turkish Cypriots about a hundred years ago. It is hoped that this paper will encourage similar studies of other communities where different large-group identities live side by side, and that such studies may support their peaceful co-existence.

  7. Investigation of a bio-inspired lift-enhancing effector on a 2D airfoil.

    PubMed

    Johnston, Joe; Gopalarathnam, Ashok

    2012-09-01

    A flap mounted on the upper surface of an airfoil, called a 'lift-enhancing effector', has been shown in wind tunnel tests to have a similar function to a bird's covert feathers, which rise off the wing's surface in response to separated flows. The effector, fabricated from a thin Mylar sheet, is allowed to rotate freely about its leading edge. The tests were performed in the NCSU subsonic wind tunnel at a chord Reynolds number of 4 × 10(5). The maximum lift coefficient with the effector was the same as that for the clean airfoil, but was maintained over an angle-of-attack range from 12° to almost 20°, resulting in a very gentle stall behavior. To better understand the aerodynamics and to estimate the deployment angle of the free-moving effector, fixed-angle effectors fabricated out of stiff wood were also tested. A progressive increase in the stall angle of attack with increasing effector angle was observed, with diminishing returns beyond the effector angle of 60°. Drag tests on both the free-moving and fixed effectors showed a marked improvement in drag at high angles of attack. Oil flow visualization on the airfoil with and without the fixed-angle effectors proved that the effector causes the separation point to move aft on the airfoil, as compared to the clean airfoil. This is thought to be the main mechanism by which an effector improves both lift and drag. A comparison of the fixed-effector results with those from the free-effector tests shows that the free effector's deployment angle is between 30° and 45°. When operating at and beyond the clean airfoil's stall angle, the free effector automatically deploys to progressively higher angles with increasing angles of attack. This slows down the rapid upstream movement of the separation point and avoids the severe reduction in the lift coefficient and an increase in the drag coefficient that are seen on the clean airfoil at the onset of stall. Thus, the effector postpones the stall by 4-8° and makes the

  8. bMERB domains are bivalent Rab8 family effectors evolved by gene duplication.

    PubMed

    Rai, Amrita; Oprisko, Anastasia; Campos, Jeremy; Fu, Yangxue; Friese, Timon; Itzen, Aymelt; Goody, Roger S; Gazdag, Emerich Mihai; Müller, Matthias P

    2016-08-23

    In their active GTP-bound form, Rab proteins interact with proteins termed effector molecules. In this study, we have thoroughly characterized a Rab effector domain that is present in proteins of the Mical and EHBP families, both known to act in endosomal trafficking. Within our study, we show that these effectors display a preference for Rab8 family proteins (Rab8, 10, 13 and 15) and that some of the effector domains can bind two Rab proteins via separate binding sites. Structural analysis allowed us to explain the specificity towards Rab8 family members and the presence of two similar Rab binding sites that must have evolved via gene duplication. This study is the first to thoroughly characterize a Rab effector protein that contains two separate Rab binding sites within a single domain, allowing Micals and EHBPs to bind two Rabs simultaneously, thus suggesting previously unknown functions of these effector molecules in endosomal trafficking.

  9. Cellular Signaling Pathways and Posttranslational Modifications Mediated by Nematode Effector Proteins1

    PubMed Central

    Hewezi, Tarek

    2015-01-01

    Plant-parasitic cyst and root-knot nematodes synthesize and secrete a suite of effector proteins into infected host cells and tissues. These effectors are the major virulence determinants mediating the transformation of normal root cells into specialized feeding structures. Compelling evidence indicates that these effectors directly hijack or manipulate refined host physiological processes to promote the successful parasitism of host plants. Here, we provide an update on recent progress in elucidating the molecular functions of nematode effectors. In particular, we emphasize how nematode effectors modify plant cell wall structure, mimic the activity of host proteins, alter auxin signaling, and subvert defense signaling and immune responses. In addition, we discuss the emerging evidence suggesting that nematode effectors target and recruit various components of host posttranslational machinery in order to perturb the host signaling networks required for immunity and to regulate their own activity and subcellular localization. PMID:26315856

  10. MIX and match: mobile T6SS MIX-effectors enhance bacterial fitness.

    PubMed

    Salomon, Dor

    2016-01-01

    Protein secretion systems that mediate interbacterial competition secret a wide repertoire of antibacterial toxins. A major player in these competitions is the newly discovered bacterial type VI secretion system (T6SS). We recently found that a subset of polymorphic MIX-effectors, which are a widespread class of effectors secreted by T6SSs, are horizontally shared between marine bacteria and are used to diversify their T6SS effector repertoires, thus enhancing their environmental fitness. In this commentary, I expand on the ideas that were introduced in the previous report, and further speculate on the possible mobility of other MIX-effectors. In addition, I discuss the possible role of horizontal gene transfer in the dissemination of MIX-effectors through bacterial genomes, as well as its possible role in diversifying the T6SS effector repertoire.

  11. Local sensory control of a dexterous end effector

    NASA Technical Reports Server (NTRS)

    Pinto, Victor H.; Everett, Louis J.; Driels, Morris

    1990-01-01

    A numerical scheme was developed to solve the inverse kinematics for a user-defined manipulator. The scheme was based on a nonlinear least-squares technique which determines the joint variables by minimizing the difference between the target end effector pose and the actual end effector pose. The scheme was adapted to a dexterous hand in which the joints are either prismatic or revolute and the fingers are considered open kinematic chains. Feasible solutions were obtained using a three-fingered dexterous hand. An algorithm to estimate the position and orientation of a pre-grasped object was also developed. The algorithm was based on triangulation using an ideal sensor and a spherical object model. By choosing the object to be a sphere, only the position of the object frame was important. Based on these simplifications, a minimum of three sensors are needed to find the position of a sphere. A two dimensional example to determine the position of a circle coordinate frame using a two-fingered dexterous hand was presented.

  12. Expression of epigenetic effectors in decidualizing human endometrial stromal cells.

    PubMed

    Grimaldi, Giulia; Christian, Mark; Quenby, Siobhan; Brosens, Jan J

    2012-09-01

    Cyclic differentiation of human endometrial stromal cells (HESCs) into decidual cells is a highly coordinated process essential for embryo implantation and pregnancy. This differentiation process is closely recapitulated in culture upon exposure of purified HESCs to cyclic AMP and progesterone signaling. Mining of gene expression data revealed that HESCs express 147 genes coding for epigenetic effectors, 33 (22%) of which are significantly regulated (P < 0.05) upon decidualization. Among these are genes encoding for histone-modifying proteins and their cofactors, histone-binding proteins, histone variants, CpG-binding proteins and DNA methyltransferases (DNMTs). Interestingly, more than two-thirds of differentially expressed chromatin-modifying genes are down-regulated upon the transition from a proliferative to a differentiated HESC phenotype. Despite the strong regulation of DNMTs, colorimetric and long interspersed nuclear element 1 methylation assays did not show global changes in DNA methylation levels upon differentiation of HESCs. Taken together, the coordinated regulation of diverse effector molecules suggests that complex epigenetic modification at specific loci underpins the acquisition of a decidual endometrial phenotype.

  13. Armet is an effector protein mediating aphid-plant interactions.

    PubMed

    Wang, Wei; Dai, Huaien; Zhang, Yi; Chandrasekar, Raman; Luo, Lan; Hiromasa, Yasuaki; Sheng, Changzhong; Peng, Gongxin; Chen, Shaoliang; Tomich, John M; Reese, John; Edwards, Owain; Kang, Le; Reeck, Gerald; Cui, Feng

    2015-05-01

    Aphid saliva is predicted to contain proteins that modulate plant defenses and facilitate feeding. Armet is a well-characterized bifunctional protein in mammalian systems. Here we report a new role of Armet, namely as an effector protein in the pea aphid, Acyrthosiphon pisum. Pea aphid Armet's physical and chemical properties and its intracellular role are comparable to those reported for mammalian Armets. Uniquely, we detected Armet in aphid watery saliva and in the phloem sap of fava beans fed on by aphids. Armet's transcript level is several times higher in the salivary gland when aphids feed on bean plants than when they feed on an artificial diet. Knockdown of the Armet transcript by RNA interference disturbs aphid feeding behavior on fava beans measured by the electrical penetration graph technique and leads to a shortened life span. Inoculation of pea aphid Armet protein into tobacco leaves induced a transcriptional response that included pathogen-responsive genes. The data suggest that Armet is an effector protein mediating aphid-plant interactions.

  14. Heat shock proteins, end effectors of myocardium ischemic preconditioning?

    PubMed Central

    Guisasola, María Concepcion; Desco, Maria del Mar; Gonzalez, Fernanda Silvana; Asensio, Fernando; Dulin, Elena; Suarez, Antonio; Garcia Barreno, Pedro

    2006-01-01

    The purpose of this study was to investigate (1) whether ischemia-reperfusion increased the content of heat shock protein 72 (Hsp72) transcripts and (2) whether myocardial content of Hsp72 is increased by ischemic preconditioning so that they can be considered as end effectors of preconditioning. Twelve male minipigs (8 protocol, 4 sham) were used, with the following ischemic preconditioning protocol: 3 ischemia and reperfusion 5-minute alternative cycles and last reperfusion cycle of 3 hours. Initial and final transmural biopsies (both in healthy and ischemic areas) were taken in all animals. Heat shock protein 72 messenger ribonucleic acid (mRNA) expression was measured by a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method using complementary DNA normalized against the housekeeping gene cyclophilin. The identification of heat shock protein 72 was performed by immunoblot. In our “classic” preconditioning model, we found no changes in mRNA hsp72 levels or heat shock protein 72 content in the myocardium after 3 hours of reperfusion. Our experimental model is valid and the experimental techniques are appropriate, but the induction of heat shock proteins 72 as end effectors of cardioprotection in ischemic preconditioning does not occur in the first hours after ischemia, but probably at least 24 hours after it, in the so-called “second protection window.” PMID:17009598

  15. Flight Control Using Distributed Shape-Change Effector Arrays

    NASA Technical Reports Server (NTRS)

    Raney, David L.; Montgomery, Raymond C.; Green, Lawrence I.; Park, Michael A.

    2000-01-01

    Recent discoveries in material science and fluidics have been used to create a variety of novel effector devices that offer great potential to enable new approaches to aerospace vehicle flight control. Examples include small inflatable blisters, shape-memory alloy diaphragms, and piezoelectric patches that may be used to produce distortions or bumps on the surface of an airfoil to generate control moments. Small jets have also been used to produce a virtual shape-change through fluidic means by creating a recirculation bubble on the surface of an airfoil. An advanced aerospace vehicle might use distributed arrays of hundreds of such devices to generate moments for stabilization and maneuver control, either augmenting or replacing conventional ailerons, flaps or rudders. This research demonstrates the design and use of shape-change device arrays for a tailless aircraft in a low-rate maneuvering application. A methodology for assessing the control authority of the device arrays is described, and a suite of arrays is used in a dynamic simulation to illustrate allocation and deployment methodologies. Although the authority of the preliminary shape-change array designs studied in this paper appeared quite low, the simulation results indicate that the effector suite possessed sufficient authority to stabilize and maneuver the vehicle in mild turbulence.

  16. Macrophages are critical effectors of antibody therapies for cancer.

    PubMed

    Weiskopf, Kipp; Weissman, Irving L

    2015-01-01

    Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients.

  17. Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter

    PubMed Central

    Sontag, Ryan L.; Nakayasu, Ernesto S.; Brown, Roslyn N.; Niemann, George S.; Sydor, Michael A.; Sanchez, Octavio; Ansong, Charles; Lu, Shao-Yeh; Choi, Hyungwon; Valleau, Dylan; Weitz, Karl K.; Savchenko, Alexei; Cambronne, Eric D.

    2016-01-01

    ABSTRACT Many pathogenic bacteria of the family Enterobacteriaceae use type III secretion systems to inject virulence proteins, termed “effectors,” into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from the Enterobacteriaceae intracellular pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium. We identified 54 high-confidence host interactors for the Salmonella effectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for the Citrobacter effectors EspT, NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfH Salmonella protein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. IMPORTANCE During infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets of Salmonella and Citrobacter effectors, which will help elucidate their mechanisms of action. PMID:27822540

  18. Evaluation of Secretion Prediction Highlights Differing Approaches Needed for Oomycete and Fungal Effectors.

    PubMed

    Sperschneider, Jana; Williams, Angela H; Hane, James K; Singh, Karam B; Taylor, Jennifer M

    2015-01-01

    The steadily increasing number of sequenced fungal and oomycete genomes has enabled detailed studies of how these eukaryotic microbes infect plants and cause devastating losses in food crops. During infection, fungal and oomycete pathogens secrete effector molecules which manipulate host plant cell processes to the pathogen's advantage. Proteinaceous effectors are synthesized intracellularly and must be externalized to interact with host cells. Computational prediction of secreted proteins from genomic sequences is an important technique to narrow down the candidate effector repertoire for subsequent experimental validation. In this study, we benchmark secretion prediction tools on experimentally validated fungal and oomycete effectors. We observe that for a set of fungal SwissProt protein sequences, SignalP 4 and the neural network predictors of SignalP 3 (D-score) and SignalP 2 perform best. For effector prediction in particular, the use of a sensitive method can be desirable to obtain the most complete candidate effector set. We show that the neural network predictors of SignalP 2 and 3, as well as TargetP were the most sensitive tools for fungal effector secretion prediction, whereas the hidden Markov model predictors of SignalP 2 and 3 were the most sensitive tools for oomycete effectors. Thus, previous versions of SignalP retain value for oomycete effector prediction, as the current version, SignalP 4, was unable to reliably predict the signal peptide of the oomycete Crinkler effectors in the test set. Our assessment of subcellular localization predictors shows that cytoplasmic effectors are often predicted as not extracellular. This limits the reliability of secretion predictions that depend on these tools. We present our assessment with a view to informing future pathogenomics studies and suggest revised pipelines for secretion prediction to obtain optimal effector predictions in fungi and oomycetes.

  19. Evaluation of Secretion Prediction Highlights Differing Approaches Needed for Oomycete and Fungal Effectors

    PubMed Central

    Sperschneider, Jana; Williams, Angela H.; Hane, James K.; Singh, Karam B.; Taylor, Jennifer M.

    2015-01-01

    The steadily increasing number of sequenced fungal and oomycete genomes has enabled detailed studies of how these eukaryotic microbes infect plants and cause devastating losses in food crops. During infection, fungal and oomycete pathogens secrete effector molecules which manipulate host plant cell processes to the pathogen's advantage. Proteinaceous effectors are synthesized intracellularly and must be externalized to interact with host cells. Computational prediction of secreted proteins from genomic sequences is an important technique to narrow down the candidate effector repertoire for subsequent experimental validation. In this study, we benchmark secretion prediction tools on experimentally validated fungal and oomycete effectors. We observe that for a set of fungal SwissProt protein sequences, SignalP 4 and the neural network predictors of SignalP 3 (D-score) and SignalP 2 perform best. For effector prediction in particular, the use of a sensitive method can be desirable to obtain the most complete candidate effector set. We show that the neural network predictors of SignalP 2 and 3, as well as TargetP were the most sensitive tools for fungal effector secretion prediction, whereas the hidden Markov model predictors of SignalP 2 and 3 were the most sensitive tools for oomycete effectors. Thus, previous versions of SignalP retain value for oomycete effector prediction, as the current version, SignalP 4, was unable to reliably predict the signal peptide of the oomycete Crinkler effectors in the test set. Our assessment of subcellular localization predictors shows that cytoplasmic effectors are often predicted as not extracellular. This limits the reliability of secretion predictions that depend on these tools. We present our assessment with a view to informing future pathogenomics studies and suggest revised pipelines for secretion prediction to obtain optimal effector predictions in fungi and oomycetes. PMID:26779196

  20. Ring the BELL and tie the KNOX: roles for TALEs in gynoecium development.

    PubMed

    Arnaud, Nicolas; Pautot, Véronique

    2014-01-01

    Carpels are leaf-like structures that bear ovules, and thus play a crucial role in the plant life cycle. In angiosperms, carpels are the last organs produced by the floral meristem and they differentiate a specialized meristematic tissue from which ovules develop. Members of the three-amino-acid-loop-extension (TALE) class of homeoproteins constitute major regulators of meristematic activity. This family contains KNOTTED-like (KNOX) and BEL1-like (BLH or BELL) homeodomain proteins, which function as heterodimers. KNOX proteins can have different BELL partners, leading to multiple combinations with distinct activities, and thus regulate many aspects of plant morphogenesis, including gynoecium development. TALE proteins act primarily through direct regulation of hormonal pathways and key transcriptional regulators. This review focuses on the contribution of TALE proteins to gynoecium development and connects TALE transcription factors to carpel gene regulatory networks.

  1. The Fairy-Folk Tale in Media Art: Reflections of Disney and Duvall.

    ERIC Educational Resources Information Center

    Molloy, Toni

    1988-01-01

    Focuses on Walt Disney and Shelley Duvall, mass media producers who furnish children with fairy-folklore. Compares and contrasts what Disney and Duvall do and do not convey through their fairy-folk tales. (MS)

  2. Tandem repeat modification during double-strand break repair induced by an engineered TAL effector nuclease in zebrafish genome.

    PubMed

    Huang, Wanxu; Zheng, Jianbo; He, Ying; Luo, Chen

    2013-01-01

    Tandem repeats (TRs) are abundant and widely distributed in eukaryotic genomes. TRs are thought to have various functions in gene transcription, DNA methylation, nucleosome position and chromatin organization. Variation of repeat units in the genome is observed in association with a number of diseases, such as Fragile X Syndrome, Huntington's disease and Friedreich's ataxia. However, the underlying mechanisms involved are poorly understood, largely owing to the technical limitations in modification of TRs at definite sites in the genome in vivo. Transcription activator-like effector nucleases (TALENs) are widely used in recent years in gene targeting for their specific binding to target sequences when engineered in vitro. Here, we show that the repair of a double-strand break (DSB) induced by TALENs adjacent to a TR can produce serial types of mutations in the TR region. Sequencing analysis revealed that there are three types of mutations induced by the DSB repair, including indels only within the TR region or within the flanking TALEN target region or simutaneously within both regions. Therefore, desired TR mutant types can be conveniently obtained by using engineered TALENs. These results demonstrate that TALENs can serve as a convenient tool for modifying TRs in the genome in studying the functions of TRs.

  3. A Plethora of Virulence Strategies Hidden Behind Nuclear Targeting of Microbial Effectors

    PubMed Central

    Rivas, Susana; Genin, Stéphane

    2011-01-01

    Plant immune responses depend on the ability to couple rapid recognition of the invading microbe to an efficient response. During evolution, plant pathogens have acquired the ability to deliver effector molecules inside host cells in order to manipulate cellular and molecular processes and establish pathogenicity. Following translocation into plant cells, microbial effectors may be addressed to different subcellular compartments. Intriguingly, a significant number of effector proteins from different pathogenic microorganisms, including viruses, oomycetes, fungi, nematodes, and bacteria, is targeted to the nucleus of host cells. In agreement with this observation, increasing evidence highlights the crucial role played by nuclear dynamics, and nucleocytoplasmic protein trafficking during a great variety of analyzed plant–pathogen interactions. Once in the nucleus, effector proteins are able to manipulate host transcription or directly subvert essential host components to promote virulence. Along these lines, it has been suggested that some effectors may affect histone packing and, thereby, chromatin configuration. In addition, microbial effectors may either directly activate transcription or target host transcription factors to alter their regular molecular functions. Alternatively, nuclear translocation of effectors may affect subcellular localization of their cognate resistance proteins in a process that is essential for resistance protein-mediated plant immunity. Here, we review recent progress in our field on the identification of microbial effectors that are targeted to the nucleus of host plant cells. In addition, we discuss different virulence strategies deployed by microbes, which have been uncovered through examination of the mechanisms that guide nuclear localization of effector proteins. PMID:22639625

  4. Filamentous pathogen effectors interfering with small RNA silencing in plant hosts.

    PubMed

    Ye, Wenwu; Ma, Wenbo

    2016-08-01

    Filamentous eukaryotic pathogens including fungi and oomycetes are major threats of plant health. During the co-evolutionary arms race with the hosts, these pathogens have evolved a large repertoire of secreted virulence proteins, called effectors, to facilitate colonization and infection. Many effectors are believed to directly manipulate targeted processes inside the host cells; and a fundamental function of the effectors is to dampen immunity. Recent evidence suggests that the destructive oomycete pathogens in the genus Phytophthora encode RNA silencing suppressors. These effectors play an important virulence role during infection, likely through their inhibitory effect on host small RNA-mediated defense.

  5. Two Fis Regulators Directly Repress the Expression of Numerous Effector-Encoding Genes in Legionella pneumophila

    PubMed Central

    Zusman, Tal; Speiser, Yariv

    2014-01-01

    Legionella pneumophila is an intracellular human pathogen that utilizes the Icm/Dot type IVB secretion system to translocate a large repertoire of effectors into host cells. For most of these effectors, there is no information regarding their regulation. Therefore, the aim of this study was to examine the involvement of the three L. pneumophila Fis homologs in the regulation of effector-encoding genes. Deletion mutants constructed in the genes encoding the three Fis regulators revealed that Fis1 (lpg0542 gene) and Fis3 (lpg1743) but not Fis2 (lpg1370) are partially required for intracellular growth of L. pneumophila in Acanthamoeba castellanii. To identify pathogenesis-related genes directly regulated by Fis, we established a novel in vivo system which resulted in the discovery of numerous effector-encoding genes directly regulated by Fis. Further examination of these genes revealed that Fis1 and Fis3 repress the level of expression of effector-encoding genes during exponential phase. Three groups of effector-encoding genes were identified: (i) effectors regulated mainly by Fis1, (ii) effectors regulated mainly by Fis3, and (iii) effectors regulated by both Fis1 and Fis3. Examination of the upstream regulatory region of all of these effector-encoding genes revealed multiple putative Fis regulatory elements, and site-directed mutagenesis confirmed that a few of these sites constitute part of a repressor binding element. Furthermore, gel mobility shift assays demonstrated the direct relation between the Fis1 and Fis3 regulators and these regulatory elements. Collectively, our results demonstrate for the first time that two of the three L. pneumophila Fis regulators directly repress the expression of Icm/Dot effector-encoding genes. PMID:25225276

  6. The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

    DOE PAGES

    Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.; ...

    2014-12-08

    Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

  7. The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

    SciTech Connect

    Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.; Tainer, John A.

    2014-12-08

    Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

  8. Gibberellin Perception by the Gibberellin Receptor and its Effector Recognition

    NASA Astrophysics Data System (ADS)

    Hakoshima, Toshio; Murase, Kohji; Hirano, Yoshinori; Sun, Tai-Ping

    Gibberellins control a diverse range of growth and developmental processes in higher plants and have been widely utilized in the agricultural industry. By binding to a nuclear receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1), gibberellins regulate gene expression by promoting degradation of the transcriptional regulator DELLA proteins. The precise manner in which GID1 discriminates and becomes activated by bioactive gibberellins for specific binding to DELLA proteins remains unclear. We present the crystal structure of a ternary complex of Arabidopsis thaliana GID1A, a bioactive gibberellin and the N-terminal DELLA domain of GAI. In this complex, GID1a occludes gibberellin in a deep binding pocket covered by its N-terminal helical switch region, which in turn interacts with the DELLA domain containing DELLA, VHYNP and LExLE motifs. Our results establish a structural model of a plant hormone receptor which is distinct from the hormone-perception mechanism and effector recognition of the known auxin receptors.

  9. Type VI secretion effectors: poisons with a purpose

    PubMed Central

    Russell, Alistair B.; Peterson, S. Brook; Mougous, Joseph D.

    2014-01-01

    The type VI secretion system (T6SS) mediates interactions between a diverse range of Gram-negative bacterial species. Recent studies have led to a drastic increase in the number of characterized T6SS effector proteins and produced a more complete and nuanced view of the adaptive significance of the system. While the system is most often implicated in antagonism, in this review we consider the case for its involvement in both antagonistic and non-antagonistic behaviors. Clarifying the roles that T6S plays in microbial communities will contribute to broader efforts to understand the importance of microbial interactions in maintaining human and environmental health, and will inform efforts to manipulate these interactions for therapeutic or environmental benefit. PMID:24384601

  10. Molecular regulation of effector and memory T cell differentiation

    PubMed Central

    Chang, John T; Wherry, E John; Goldrath, Ananda W

    2015-01-01

    Immunological memory is a cardinal feature of adaptive immunity and an important goal of vaccination strategies. Here we highlight advances in the understanding of the diverse T lymphocyte subsets that provide acute and long-term protection from infection. These include new insights into the transcription factors, and the upstream ‘pioneering’ factors that regulate their accessibility to key sites of gene regulation, as well as metabolic regulators that contribute to the differentiation of effector and memory subsets; ontogeny and defining characteristics of tissue-resident memory lymphocytes; and origins of the remarkable heterogeneity exhibited by activated T cells. Collectively, these findings underscore progress in delineating the underlying pathways that control diversification in T cell responses but also reveal gaps in the knowledge, as well as the challenges that arise in the application of this knowledge to rationally elicit desired T cell responses through vaccination and immunotherapy. PMID:25396352

  11. End effectors and attachments for buried waste excavation equipment

    SciTech Connect

    King, R.H.

    1993-09-01

    The Buried Waste Integrated Demonstration (BWID) supports the applied research, development, demonstration, and evaluation of a suite of advanced technologies that form a comprehensive remediation system for the effective and efficient remediation of buried waste. Their efforts are identified and coordinated in support of the U.S. Department of Energy (DOE), Environmental Restoration and Waste Management (ER&WM) Department`s needs and objectives. The present focus of BWID is to support retrieval and ex-situ treatment configuration options. Future activities will explore and support containment, and stabilization efforts in addition to the retrieval/ex situ treatment options. This report presents a literature search on the state-of-the-art in end effectors and attachments in support of excavator of buried transuranic waste. Included in the report are excavator platforms and a discussion of the various attachments. Also included is it list of vendors and specifications.

  12. Direct targeting of Rab-GTPase-effector interactions.

    PubMed

    Spiegel, Jochen; Cromm, Philipp M; Itzen, Aymelt; Goody, Roger S; Grossmann, Tom N; Waldmann, Herbert

    2014-02-24

    Small GTPases are molecular switches using GDP/GTP alternation to control numerous vital cellular processes. Although aberrant function and regulation of GTPases are implicated in various human diseases, direct targeting of this class of proteins has proven difficult, as GTPase signaling and regulation is mediated by extensive and shallow protein interfaces. Here we report the development of inhibitors of protein-protein interactions involving Rab proteins, a subfamily of GTPases, which are key regulators of vesicular transport. Hydrocarbon-stapled peptides were designed based on crystal structures of Rab proteins bound to their interaction partners. These modified peptides exhibit significantly increased affinities and include a stapled peptide (StRIP3) that selectively binds to activated Rab8a and inhibits a Rab8a-effector interaction in vitro.

  13. NOD-like receptor cooperativity in effector-triggered immunity.

    PubMed

    Griebel, Thomas; Maekawa, Takaki; Parker, Jane E

    2014-11-01

    Intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are basic elements of innate immunity in plants and animals. Whereas animal NLRs react to conserved microbe- or damage-associated molecular patterns, plant NLRs intercept the actions of diverse pathogen virulence factors (effectors). In this review, we discuss recent genetic and molecular evidence for functional NLR pairs, and discuss the significance of NLR self-association and heteromeric NLR assemblies in the triggering of downstream signaling pathways. We highlight the versatility and impact of cooperating NLR pairs that combine pathogen sensing with the initiation of defense signaling in both plant and animal immunity. We propose that different NLR receptor molecular configurations provide opportunities for fine-tuning resistance pathways and enhancing the host's pathogen recognition spectrum to keep pace with rapidly evolving microbial populations.

  14. Intervention of Phytohormone Pathways by Pathogen Effectors[OPEN

    PubMed Central

    Kazan, Kemal; Lyons, Rebecca

    2014-01-01

    The constant struggle between plants and microbes has driven the evolution of multiple defense strategies in the host as well as offense strategies in the pathogen. To defend themselves from pathogen attack, plants often rely on elaborate signaling networks regulated by phytohormones. In turn, pathogens have adopted innovative strategies to manipulate phytohormone-regulated defenses. Tactics frequently employed by plant pathogens involve hijacking, evading, or disrupting hormone signaling pathways and/or crosstalk. As reviewed here, this is achieved mechanistically via pathogen-derived molecules known as effectors, which target phytohormone receptors, transcriptional activators and repressors, and other components of phytohormone signaling in the host plant. Herbivores and sap-sucking insects employ obligate pathogens such as viruses, phytoplasma, or symbiotic bacteria to intervene with phytohormone-regulated defenses. Overall, an improved understanding of phytohormone intervention strategies employed by pests and pathogens during their interactions with plants will ultimately lead to the development of new crop protection strategies. PMID:24920334

  15. Electroporation of functional bacterial effectors into mammalian cells.

    PubMed

    Sontag, Ryan L; Mihai, Cosmin; Orr, Galya; Savchenko, Alexei; Skarina, Tatiana; Cui, Hong; Cort, John R; Adkins, Joshua N; Brown, Roslyn N

    2015-01-19

    The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high

  16. Macroautophagy regulates energy metabolism during effector T cell activation.

    PubMed

    Hubbard, Vanessa M; Valdor, Rut; Patel, Bindi; Singh, Rajat; Cuervo, Ana Maria; Macian, Fernando

    2010-12-15

    Macroautophagy is a highly conserved mechanism of lysosomal-mediated protein degradation that plays a key role in maintaining cellular homeostasis by recycling amino acids, reducing the amount of damaged proteins, and regulating protein levels in response to extracellular signals. We have found that macroautophagy is induced after effector T cell activation. Engagement of the TCR and CD28 results in enhanced microtubule-associated protein 1 light chain 3 (LC3) processing, increased numbers of LC3-containing vesicles, and increased LC3 flux, indicating active autophagosome formation and clearance. The autophagosomes formed in stimulated T cells actively fuse with lysosomes to degrade their cargo. Using a conditional KO mouse model where Atg7, a critical gene for macroautophagy, is specifically deleted in T cells, we have found that macroautophagy-deficient effector Th cells have defective IL-2 and IFN-γ production and reduced proliferation after stimulation, with no significant increase in apoptosis. We have found that ATP generation is decreased when autophagy is blocked, and defects in activation-induced cytokine production are restored when an exogenous energy source is added to macroautophagy-deficient T cells. Furthermore, we present evidence showing that the nature of the cargo inside autophagic vesicles found in resting T cells differs from the cargo of autophagosomes in activated T cells, where mitochondria and other organelles are selectively excluded. These results suggest that macroautophagy is an actively regulated process in T cells that can be induced in response to TCR engagement to accommodate the bioenergetic requirements of activated T cells.

  17. Pointing Hand Stimuli Induce Spatial Compatibility Effects and Effector Priming

    PubMed Central

    Nishimura, Akio; Michimata, Chikashi

    2013-01-01

    The present study investigated the automatic influence of perceiving a picture that indicates other’s action on one’s own task performance in terms of spatial compatibility and effector priming. Participants pressed left and right buttons with their left and right hands respectively, depending on the color of a central dot target. Preceding the target, a left or right hand stimulus (pointing either to the left or right with the index or little finger) was presented. In Experiment 1, with brief presentation of the pointing hand, a spatial compatibility effect was observed: responses were faster when the direction of the pointed finger and the response position were spatially congruent than when incongruent. The spatial compatibility effect was larger for the pointing index finger stimulus compared to the pointing little finger stimulus. Experiment 2 employed longer duration of the pointing hand stimuli. In addition to the spatial compatibility effect for the pointing index finger, the effector priming effect was observed: responses were faster when the anatomical left/right identity of the pointing and response hands matched than when the pointing and response hands differed in left/right identity. The results indicate that with sufficient processing time, both spatial/symbolic and anatomical features of a static body part implying another’s action simultaneously influence different aspects of the perceiver’s own action. Hierarchical coding, according to which an anatomical code is used only when a spatial code is unavailable, may not be applicable if stimuli as well as responses contain anatomical features. PMID:23637688

  18. Protection after stroke: cellular effectors of neurovascular unit integrity

    PubMed Central

    Posada-Duque, Rafael Andres; Barreto, George E.; Cardona-Gomez, Gloria Patricia

    2014-01-01

    Neurological disorders are prevalent worldwide. Cerebrovascular diseases (CVDs), which account for 55% of all neurological diseases, are the leading cause of permanent disability, cognitive and motor disorders and dementia. Stroke affects the function and structure of blood-brain barrier, the loss of cerebral blood flow regulation, oxidative stress, inflammation and the loss of neural connections. Currently, no gold standard treatments are available outside the acute therapeutic window to improve outcome in stroke patients. Some promising candidate targets have been identified for the improvement of long-term recovery after stroke, such as Rho GTPases, cell adhesion proteins, kinases, and phosphatases. Previous studies by our lab indicated that Rho GTPases (Rac and RhoA) are involved in both tissue damage and survival, as these proteins are essential for the morphology and movement of neurons, astrocytes and endothelial cells, thus playing a critical role in the balance between cell survival and death. Treatment with a pharmacological inhibitor of RhoA/ROCK blocks the activation of the neurodegeneration cascade. In addition, Rac and synaptic adhesion proteins (p120 catenin and N-catenin) play critical roles in protection against cerebral infarction and in recovery by supporting the neurovascular unit and cytoskeletal remodeling activity to maintain the integrity of the brain parenchyma. Interestingly, neuroprotective agents, such as atorvastatin, and CDK5 silencing after cerebral ischemia and in a glutamate-induced excitotoxicity model may act on the same cellular effectors to recover neurovascular unit integrity. Therefore, future efforts must focus on individually targeting the structural and functional roles of each effector of neurovascular unit and the interactions in neural and non-neural cells in the post-ischemic brain and address how to promote the recovery or prevent the loss of homeostasis in the short, medium and long term. PMID:25177270

  19. Electroporation of Functional Bacterial Effectors into Mammalian Cells

    PubMed Central

    Sontag, Ryan L.; Mihai, Cosmin; Orr, Galya; Savchenko, Alexei; Skarina, Tatiana; Cui, Hong; Cort, John R.; Adkins, Joshua N.; Brown, Roslyn N.

    2015-01-01

    The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high

  20. Comparative reactivity of human IgE to cynomolgus monkey and human effector cells and effects on IgE effector cell potency

    PubMed Central

    Saul, Louise; Saul, Louise; Josephs, Debra H; Josephs, Debra H; Cutler, Keith; Cutler, Keith; Bradwell, Andrew; Bradwell, Andrew; Karagiannis, Panagiotis; Karagiannis, Panagiotis; Selkirk, Chris; Selkirk, Chris; Gould, Hannah J; Gould, Hannah J; Jones, Paul; Jones, Paul; Spicer, James F; Spicer, James F; Karagiannis, Sophia N; Karagiannis, Sophia N

    2014-01-01

    Background: Due to genetic similarities with humans, primates of the macaque genus such as the cynomolgus monkey are often chosen as models for toxicology studies of antibody therapies. IgE therapeutics in development depend upon engagement with the FcεRI and FcεRII receptors on immune effector cells for their function. Only limited knowledge of the primate IgE immune system is available to inform the choice of models for mechanistic and safety evaluations.   Methods: The recognition of human IgE by peripheral blood lymphocytes from cynomolgus monkey and man was compared. We used effector cells from each species in ex vivo affinity, dose-response, antibody-receptor dissociation and potency assays. Results: We report cross-reactivity of human IgE Fc with cynomolgus monkey cells, and comparable binding kinetics to peripheral blood lymphocytes from both species. In competition and dissociation assays, however, human IgE dissociated faster from cynomolgus monkey compared with human effector cells. Differences in association and dissociation kinetics were reflected in effector cell potency assays of IgE-mediated target cell killing, with higher concentrations of human IgE needed to elicit effector response in the cynomolgus monkey system. Additionally, human IgE binding on immune effector cells yielded significantly different cytokine release profiles in each species. Conclusion: These data suggest that human IgE binds with different characteristics to human and cynomolgus monkey IgE effector cells. This is likely to affect the potency of IgE effector functions in these two species, and so has relevance for the selection of biologically-relevant model systems when designing pre-clinical toxicology and functional studies. PMID:24492303

  1. The putative Agrobacterium transcriptional activator-like virulence protein VirD5 may target T-complex to prevent the degradation of coat proteins in the plant cell nucleus.

    PubMed

    Wang, Yafei; Peng, Wei; Zhou, Xu; Huang, Fei; Shao, Lingyun; Luo, Meizhong

    2014-09-01

    Agrobacterium exports at least five virulence proteins (VirE2, VirE3, VirF, VirD2, VirD5) into host cells and hijacks some host plant factors to facilitate its transformation process. Random DNA binding selection assays (RDSAs), electrophoretic mobility shift assays (EMSAs) and yeast one-hybrid systems were used to identify protein-bound DNA elements. Bimolecular fluorescence complementation, glutathione S-transferase pull-down and yeast two-hybrid assays were used to detect protein interactions. Protoplast transformation, coprecipitation, competitive binding and cell-free degradation assays were used to analyze the relationships among proteins. We found that Agrobacterium VirD5 exhibits transcriptional activation activity in yeast, is located in the plant cell nucleus, and forms homodimers. A specific VirD5-bound DNA element designated D5RE (VirD5 response element) was identified. VirD5 interacted directly with Arabidopsis VirE2 Interacting Protein 1 (AtVIP1). However, the ternary complex of VirD5-AtVIP1-VirE2 could be detected, whereas that of VirD5-AtVIP1-VBF (AtVIP1 Binding F-box protein) could not. We demonstrated that VirD5 competes with VBF for binding to AtVIP1 and stabilizes AtVIP1 and VirE2 in the cell-free degradation system. Our results indicated that VirD5 may act as both a transcriptional activator-like effector to regulate host gene expression and a protector preventing the coat proteins of the T-complex from being quickly degraded by the host's ubiquitin proteasome system (UPS).

  2. Microbe-independent entry of oomycete RxLR effectors and fungal RxLR-like effectors into plant and animal cells is specific and reproducible.

    PubMed

    Tyler, Brett M; Kale, Shiv D; Wang, Qunqing; Tao, Kai; Clark, Helen R; Drews, Kelly; Antignani, Vincenzo; Rumore, Amanda; Hayes, Tristan; Plett, Jonathan M; Fudal, Isabelle; Gu, Biao; Chen, Qinghe; Affeldt, Katharyn J; Berthier, Erwin; Fischer, Gregory J; Dou, Daolong; Shan, Weixing; Keller, Nancy P; Martin, Francis; Rouxel, Thierry; Lawrence, Christopher B

    2013-06-01

    A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, at levels well above background nonspecific entry. We also summarize host cell entry experiments with a wide diversity of oomycete and fungal effectors with RxLR or RxLR-like motifs that have been independently carried out by the seven different labs that coauthored this letter. Finally we discuss possible technical reasons why specific cell entry may have been not detected by Wawra et al. (2013).

  3. Structural evolution of differential amino acid effector regulation in plant chorismate mutases.

    PubMed

    Westfall, Corey S; Xu, Ang; Jez, Joseph M

    2014-10-10

    Chorismate mutase converts chorismate into prephenate for aromatic amino acid biosynthesis. To understand the molecular basis of allosteric regulation in the plant chorismate mutases, we analyzed the three Arabidopsis thaliana chorismate mutase isoforms (AtCM1-3) and determined the x-ray crystal structures of AtCM1 in complex with phenylalanine and tyrosine. Functional analyses show a wider range of effector control in the Arabidopsis chorismate mutases than previously reported. AtCM1 is activated by tryptophan with phenylalanine and tyrosine acting as negative effectors; however, tryptophan, cysteine, and histidine activate AtCM3. AtCM2 is a nonallosteric form. The crystal structure of AtCM1 in complex with tyrosine and phenylalanine identifies differences in the effector sites of the allosterically regulated yeast enzyme and the other two Arabidopsis isoforms. Site-directed mutagenesis of residues in the effector site reveals key features leading to differential effector regulation in these enzymes. In AtCM1, mutations of Gly-213 abolish allosteric regulation, as observed in AtCM2. A second effector site position, Gly-149 in AtCM1 and Asp-132 in AtCM3, controls amino acid effector specificity in AtCM1 and AtCM3. Comparisons of chorismate mutases from multiple plants suggest that subtle differences in the effector site are conserved in different lineages and may lead to specialized regulation of this branch point enzyme.

  4. Putative rust fungal effector proteins in infected bean and soybean leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The plant pathogenic fungi Uromyces appendiculatus and Phakopsora pachyrhizi cause debilitating rust diseases on common bean and soybean. These rust fungi secrete effector proteins that allow them to infect plants, but the effector repertoire for U. appendiculatus and P. pachyrhizi is not fully def...

  5. Differential expression of candidate salivary effector proteins in field collections of Hessian fly, Mayetiola destructor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Evidence is emerging that proteins secreted by gall forming plant-parasites are the effectors responsible for systemic changes in the host plant, such as galling and nutrient tissue formation. A large number of secreted salivary gland proteins (SSGPs) that are hypothesized to be the effectors respon...

  6. Homologous RXLR effectors from Hyaloperonospora arabidopsidis and Phytophthora sojae suppress immunity in distantly related plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. Oomycete pathogens contain very large complements of predicted effector genes defined by an RXLR host cell entry motif. The genome of Hyaloperonospora arabidopsidis (Hpa, downy mildew of Arabidopsis) ...

  7. Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants.

    PubMed

    Aung, Kyaw; Xin, Xiufang; Mecey, Christy; He, Sheng Yang

    2017-01-01

    Animal and plant pathogenic bacteria use type III secretion systems to translocate proteinaceous effectors to subvert innate immunity of their host organisms. Type III secretion/effector systems are a crucial pathogenicity factor in many bacterial pathogens of plants and animals. Pseudomonas syringae pv. tomato (Pst) DC3000 injects a total of 36 protein effectors that target a variety of host proteins. Studies of a subset of Pst DC3000 effectors demonstrated that bacterial effectors, once inside the host cell, are localized to different subcellular compartments, including plasma membrane, cytoplasm, mitochondria, chloroplast, and Trans-Golgi network, to carry out their virulence functions. Identifying the subcellular localization of bacterial effector proteins in host cells could provide substantial clues to understanding the molecular and cellular basis of the virulence activities of effector proteins. In this chapter, we present methods for transient or stable expression of bacterial effector proteins in tobacco and/or Arabidopsis thaliana for live cell imaging as well as confirming the subcellular localization in plants using fluorescent organelle markers or chemical treatment.

  8. Molecular determinants of resistance activation and suppression by Phytophthora infestans effector IPI-O

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The potato late blight pathogen, Phytophthora infestans, is able to rapidly evolve to overcome resistance genes. The pathogen accomplishes this by secreting an arsenal of proteins, termed effectors, that function to modify host cells. Although hundreds of candidate effectors have been identified in ...

  9. Visual processing at goal and effector locations is dynamically enhanced during motor preparation.

    PubMed

    Mason, Luke; Linnell, Karina J; Davis, Rob; Van Velzen, José

    2015-08-15

    Previous theoretical and experimental works has shown that preparing to act causes enhanced perceptual processing at movement-relevant locations. Up until now, this has focused almost exclusively on the goal of an action, neglecting the role of the effector. We addressed this by measuring changes in visual processing across time during motor preparation at both goal and effector locations. We compared event related potentials (ERPs) elicited by task-irrelevant visual probe stimuli at both goal and effector locations during motor preparation. Participants were instructed to place their hands on two starting positions (effector locations) and an auditory tone instructed them to immediately move to one of two target buttons (goal locations). Probe stimuli were presented in the interval between the offset of the cue and the execution of the movement at either a goal or an effector location. Probes were presented randomly at either 100ms, 200ms or 300ms after the auditory cue. Analysis of the visual N1 ERP showed enhanced visual processing at moving vs. not-moving goal locations across all three SOAs. At effector locations, enhanced processing for the moving vs. not-moving effector was only observed during the middle (200ms) SOA. These results demonstrate, for the first time, simultaneous perceptual enhancement of goal and effector locations during motor preparation. We interpret these results as reflecting a temporally and spatially specific dynamic attentional map of the environment that adapts to maximise efficiency of movement by selectively weighting processing of multiple functional components of action in parallel.

  10. A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach.

    PubMed

    Russell, Alistair B; Singh, Pragya; Brittnacher, Mitchell; Bui, Nhat Khai; Hood, Rachel D; Carl, Mike A; Agnello, Danielle M; Schwarz, Sandra; Goodlett, David R; Vollmer, Waldemar; Mougous, Joseph D

    2012-05-17

    Sophisticated mechanisms are employed to facilitate information exchange between interfacing bacteria. A type VI secretion system (T6SS) of Pseudomonas aeruginosa was shown to deliver cell wall-targeting effectors to neighboring cells. However, the generality of bacteriolytic effectors and, moreover, of antibacterial T6S remained unknown. Using parameters derived from experimentally validated bacterial T6SS effectors we identified a phylogenetically disperse superfamily of T6SS-associated peptidoglycan-degrading effectors. The effectors separate into four families composed of peptidoglycan amidase enzymes of differing specificities. Effectors strictly co-occur with cognate immunity proteins, indicating that self-intoxication is a general property of antibacterial T6SSs and effector delivery by the system exerts a strong selective pressure in nature. The presence of antibacterial effectors in a plethora of organisms, including many that inhabit or infect polymicrobial niches in the human body, suggests that the system could mediate interbacterial interactions of both environmental and clinical significance.

  11. Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv. campestris.

    PubMed

    Jiang, Guo-Feng; Jiang, Bo-Le; Yang, Mei; Liu, San; Liu, Jiao; Liang, Xiao-Xia; Bai, Xian-Fang; Tang, Dong-Jie; Lu, Guang-Tao; He, Yong-Qiang; Yu, Di-Qiu; Tang, Ji-Liang

    2013-01-01

    It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.

  12. Elucidating the Role of Effectors in Plant-Fungal Interactions: Progress and Challenges

    PubMed Central

    Selin, Carrie; de Kievit, Teresa R.; Belmonte, Mark F.; Fernando, W. G. Dilantha

    2016-01-01

    Pathogenic fungi have diverse growth lifestyles that support fungal colonization on plants. Successful colonization and infection for all lifestyles depends upon the ability to modify living host plants to sequester the necessary nutrients required for growth and reproduction. Secretion of virulence determinants referred to as “effectors” is assumed to be the key governing factor that determines host infection and colonization. Effector proteins are capable of suppressing plant defense responses and alter plant physiology to accommodate fungal invaders. This review focuses on effector molecules of biotrophic and hemibiotrophic plant pathogenic fungi, and the mechanism required for the release and uptake of effector molecules by the fungi and plant cells, respectively. We also place emphasis on the discovery of effectors, difficulties associated with predicting the effector repertoire, and fungal genomic features that have helped promote effector diversity leading to fungal evolution. We discuss the role of specific effectors found in biotrophic and hemibiotrophic fungi and examine how CRISPR/Cas9 technology may provide a new avenue for accelerating our ability in the discovery of fungal effector function. PMID:27199930

  13. Structure of NS1A effector domain from the influenza A/Udorn/72 virus

    SciTech Connect

    Xia, Shuangluo; Monzingo, Arthur F.; Robertus, Jon D.

    2009-01-01

    The structure of the effector domain of the influenza protein NS1, a validated antiviral drug target, has been solved in two space groups. The nonstructural protein NS1A from influenza virus is a multifunctional virulence factor and a potent inhibitor of host immunity. It has two functional domains: an N-terminal 73-amino-acid RNA-binding domain and a C-terminal effector domain. Here, the crystallographic structure of the NS1A effector domain of influenza A/Udorn/72 virus is presented. Structure comparison with the NS1 effector domain from mouse-adapted influenza A/Puerto Rico/8/34 (PR8) virus strain reveals a similar monomer conformation but a different dimer interface. Further analysis and evaluation shows that the dimer interface observed in the structure of the PR8 NS1 effector domain is likely to be a crystallographic packing effect. A hypothetical model of the intact NS1 dimer is presented.

  14. Legionella pneumophila, armed to the hilt: justifying the largest arsenal of effectors in the bacterial world.

    PubMed

    Ensminger, Alexander W

    2016-02-01

    Many bacterial pathogens use dedicated translocation systems to deliver arsenals of effector proteins to their hosts. Once inside the host cytosol, these effectors modulate eukaryotic cell biology to acquire nutrients, block microbial degradation, subvert host defenses, and enable pathogen transmission to other hosts. Among all bacterial pathogens studied to date, the gram-negative pathogen, Legionella pneumophila, maintains the largest arsenal of effectors, with over 330 effector proteins translocated by the Dot/Icm type IVB translocation system. In this review, I will discuss some of the recent work on understanding the consequences of this large arsenal. I will also present several models that seek to explain how L. pneumophila has acquired and subsequently maintained so many more effectors than its peers.

  15. [Th17 cells, a novel proinflammatory effector CD4 T cell population].

    PubMed

    Leung-Theung-Long, Stéphane; Guerder, Sylvie

    2008-11-01

    After more than 20 years of hegemony, the Th1-Th2 paradigm was recently shaken by the discovery of a novel population of CD4 effector T cells, the Th17 cells. Th17 effector cells produce IL-17 and IL-22 and thus have pro-inflammatory properties notably favoring neutrophils recruitment and thus control of extracellular bacteria mainly at the epithelium surface. Th17 cells appear also as the major inducer of organ specific autoimmune pathologies such as EAE or rheumatoid arthritis, a function previously attributed to Th1 effector cells. The discovery of Th17 cells further supports the notion that effector CD4 T cells responses are diverse in vivo and that fine tuning of these different effector cells is critical to maintain tissue integrity.

  16. Coyote & Little Turtle = Iisaw Niqw Yongosonhoya: A Traditional Hopi Tale and Coyote & the Winnowing Birds = Iisaw Niqw Tsaayantotaqam Tsiroot: A Traditional Hopi Tale. Original Language Series.

    ERIC Educational Resources Information Center

    Sekaquaptewa, Emory, Ed.; Pepper, Barbara, Ed.

    Intended to promote the preservation of the Hopi language, two illustrated children's books present traditional Hopi tales in bilingual format. Based on a story told by Herschel Talashoema, "Coyote & Little Turtle" tells how Little Turtle tricked Coyote into carrying him from the hot sand that burned his feet to Little Turtle's home in a spring.…

  17. Ancient DNA and the tropics: a rodent's tale

    PubMed Central

    Gutiérrez-García, Tania A.; Vázquez-Domínguez, Ella; Arroyo-Cabrales, Joaquín; Kuch, Melanie; Enk, Jacob; King, Christine; Poinar, Hendrik N.

    2014-01-01

    Most genetic studies of Holocene fauna have been performed with ancient samples from dry and cold regions, in which preservation of fossils is facilitated and molecular damage is reduced. Ancient DNA work from tropical regions has been precluded owing to factors that limit DNA preservation (e.g. temperature, hydrolytic damage). We analysed ancient DNA from rodent jawbones identified as Ototylomys phyllotis, found in Holocene and Late Pleistocene stratigraphic layers from Loltún, a humid tropical cave located in the Yucatan peninsula. We extracted DNA and amplified six short overlapping fragments of the cytochrome b gene, totalling 666 bp, which represents an unprecedented success considering tropical ancient DNA samples. We performed genetic, phylogenetic and divergence time analyses, combining sequences from ancient and modern O. phyllotis, in order to assess the ancestry of the Loltún samples. Results show that all ancient samples fall into a unique clade that diverged prior to the divergence of the modern O. phyllotis, supporting it as a distinct Pleistocene form of the Ototylomys genus. Hence, this rodent's tale suggests that the sister group to modern O. phyllotis arose during the Miocene–Pliocene, diversified during the Pleistocene and went extinct in the Holocene. PMID:24899682

  18. Horror films: tales to master terror or shapers of trauma?

    PubMed

    Ballon, Bruce; Leszcz, Molyn

    2007-01-01

    The authors review the literature of cinematic-related psychiatric case reports and report the case of a 22-year-old woman who presented with intrusive thoughts of demonic possession and flashbacks of the film The Exorcist. Cinematic neurosis may be considered a form of psychological crisis shaped by exposure to a film narrative that is emotionally and culturally significant to the individual. The structure of horror films are examined from the perspectives of trauma theory, narrative theory, and borderline personality organization theories, using the film The Exorcist as an example. Within this framework, the horror film can be seen as a cultural tale that provides a mechanism for attempting mastery over anxieties involving issues of separation, loss, autonomy, and identity. An individual will identify with narrative elements that resonate in personal life experiences and cultural factors embedded within the film, which carry levels of either stress that will be mastered, or act as a trauma to the viewer. The outcome of this exposure is related to how the individual's personality structure is organized in combination with the stresses they are experiencing.

  19. A Tale of Two Comets - The FORCAST Story

    NASA Astrophysics Data System (ADS)

    Woodward, Charles

    2015-10-01

    The properties of small, primitive bodies in the solar system, including individual comets and comet-families, whose origins lie beyond the water frost line (> 5 AU) provide critical insight into the formation of Solar System solids and establishes observational constraints for planetary system formation invoking migration. We propose to obtain 4.9 to 37.1 micron spectra and four-filter imagery of two Oort Cloud comets, C/2013 US10 (Catalina) and C/2013 X1 (Pan-STARRS), with FORCAST to determine the composition of the coma materials. Cometary dust contains remnants of both primordial material in the comet-formation zone as well as processed material transported from hotter regions closer to the Sun. We will address two questions: Q1. What information do cometary grains provide us concerning the evolution of the early solar system? Q2. What are the fundamental differences between comets originating from different regions and epochs during solar system formation? Comets form our most direct link to the earliest stages of the formation and evolution of the solar system. The narrative tale, gleaned through the study of grains, ices, and volatiles, is an account of our origins.

  20. Ancient DNA and the tropics: a rodent's tale.

    PubMed

    Gutiérrez-García, Tania A; Vázquez-Domínguez, Ella; Arroyo-Cabrales, Joaquín; Kuch, Melanie; Enk, Jacob; King, Christine; Poinar, Hendrik N

    2014-06-01

    Most genetic studies of Holocene fauna have been performed with ancient samples from dry and cold regions, in which preservation of fossils is facilitated and molecular damage is reduced. Ancient DNA work from tropical regions has been precluded owing to factors that limit DNA preservation (e.g. temperature, hydrolytic damage). We analysed ancient DNA from rodent jawbones identified as Ototylomys phyllotis, found in Holocene and Late Pleistocene stratigraphic layers from Loltún, a humid tropical cave located in the Yucatan peninsula. We extracted DNA and amplified six short overlapping fragments of the cytochrome b gene, totalling 666 bp, which represents an unprecedented success considering tropical ancient DNA samples. We performed genetic, phylogenetic and divergence time analyses, combining sequences from ancient and modern O. phyllotis, in order to assess the ancestry of the Loltún samples. Results show that all ancient samples fall into a unique clade that diverged prior to the divergence of the modern O. phyllotis, supporting it as a distinct Pleistocene form of the Ototylomys genus. Hence, this rodent's tale suggests that the sister group to modern O. phyllotis arose during the Miocene-Pliocene, diversified during the Pleistocene and went extinct in the Holocene.

  1. The genome sequence and effector complement of the flax rust pathogen Melampsora lini

    PubMed Central

    Nemri, Adnane; Saunders, Diane G. O.; Anderson, Claire; Upadhyaya, Narayana M.; Win, Joe; Lawrence, Gregory J.; Jones, David A.; Kamoun, Sophien; Ellis, Jeffrey G.; Dodds, Peter N.

    2014-01-01

    Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed most extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimize parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analyzed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their

  2. Using Hierarchical Clustering of Secreted Protein Families to Classify and Rank Candidate Effectors of Rust Fungi

    PubMed Central

    Saunders, Diane G. O.; Win, Joe; Cano, Liliana M.; Szabo, Les J.; Kamoun, Sophien; Raffaele, Sylvain

    2012-01-01

    Rust fungi are obligate biotrophic pathogens that cause considerable damage on crop plants. Puccinia graminis f. sp. tritici, the causal agent of wheat stem rust, and Melampsora larici-populina, the poplar leaf rust pathogen, have strong deleterious impacts on wheat and poplar wood production, respectively. Filamentous pathogens such as rust fungi secrete molecules called disease effectors that act as modulators of host cell physiology and can suppress or trigger host immunity. Current knowledge on effectors from other filamentous plant pathogens can be exploited for the characterisation of effectors in the genome of recently sequenced rust fungi. We designed a comprehensive in silico analysis pipeline to identify the putative effector repertoire from the genome of two plant pathogenic rust fungi. The pipeline is based on the observation that known effector proteins from filamentous pathogens have at least one of the following properties: (i) contain a secretion signal, (ii) are encoded by in planta induced genes, (iii) have similarity to haustorial proteins, (iv) are small and cysteine rich, (v) contain a known effector motif or a nuclear localization signal, (vi) are encoded by genes with long intergenic regions, (vii) contain internal repeats, and (viii) do not contain PFAM domains, except those associated with pathogenicity. We used Markov clustering and hierarchical clustering to classify protein families of rust pathogens and rank them according to their likelihood of being effectors. Using this approach, we identified eight families of candidate effectors that we consider of high value for functional characterization. This study revealed a diverse set of candidate effectors, including families of haustorial expressed secreted proteins and small cysteine-rich proteins. This comprehensive classification of candidate effectors from these devastating rust pathogens is an initial step towards probing plant germplasm for novel resistance components. PMID:22238666

  3. Diverse Secreted Effectors Are Required for Salmonella Persistence in a Mouse Infection Model

    SciTech Connect

    Kidwai, Afshan S.; Mushamiri, Ivy T.; Niemann, George; Brown, Roslyn N.; Adkins, Joshua N.; Heffron, Fred

    2013-08-12

    Salmonella enterica serovar Typhimurium causes typhoid-like disease in mice and is a model of typhoid fever in humans. One of the hallmarks of typhoid is persistence, the ability of the bacteria to survive in the host weeks after infection. Virulence factors called effectors facilitate this process by direct transfer to the cytoplasm of infected cells thereby subverting cellular processes. Secretion of effectors to the cell cytoplasm takes place through multiple routes, including two separate type III secretion (T3SS) apparati as well as outer membrane vesicles. The two T3SS are encoded on separate pathogenicity islands, SPI-1 and -2, with SPI-1 more strongly associated with the intestinal phase of infection, and SPI-2 with the systemic phase. Both T3SS are required for persistence, but the effectors required have not been systematically evaluated. In this study, mutations in 48 described effectors were tested for persistence. We replaced each effector with a specific DNA barcode sequence by allelic exchange and co-infected with a wild-type reference to calculate the ratio of wild-type parent to mutant at different times after infection. The competitive index (CI) was determined by quantitative PCR in which primers that correspond to the barcode were used for amplification. Mutations in all but seven effectors reduced persistence demonstrating that most effectors were required. One exception was CigR, a recently discovered effector that is widely conserved throughout enteric bacteria. Deletion of cigR increased lethality, suggesting that it may be an anti-virulence factor. The fact that almost all Salmonella effectors are required for persistence argues against redundant functions. This is different from effector repertoires in other intracellular pathogens such as Legionella.

  4. At the Frontier; RXLR Effectors Crossing the Phytophthora–Host Interface

    PubMed Central

    Bouwmeester, Klaas; Meijer, Harold J. G.; Govers, Francine

    2011-01-01

    Plants are constantly beset by pathogenic organisms. To successfully infect their hosts, plant pathogens secrete effector proteins, many of which are translocated to the inside of the host cell where they manipulate normal physiological processes and undermine host defense. The way by which effectors cross the frontier to reach the inside of the host cell varies among different classes of pathogens. For oomycete plant pathogens – like the potato late blight pathogen Phytophthora infestans – it has been shown that effector translocation to the host cell cytoplasm is dependent on conserved amino acid motifs that are present in the N-terminal part of effector proteins. One of these motifs, known as the RXLR motif, has a strong resemblance with a host translocation motif found in effectors secreted by Plasmodium species. These malaria parasites, that reside inside specialized vacuoles in red blood cells, make use of a specific protein translocation complex to export effectors from the vacuole into the red blood cell. Whether or not also oomycete RXLR effectors require a translocation complex to cross the frontier is still under investigation. For one P. infestans RXLR effector named IPI-O we have found a potential host target that could play a role in establishing the first contact between this effector and the host cell. This membrane spanning lectin receptor kinase, LecRK-I.9, interacts with IPI-O via the tripeptide RGD that overlaps with the RXLR motif. In animals, RGD is a well-known cell adhesion motif; it binds to integrins, which are membrane receptors that regulate many cellular processes and which can be hijacked by pathogens for either effector translocation or pathogen entry into host cells. PMID:22645549

  5. Effector-repressor interactions, binding of a single effector molecule to the operator-bound TtgR homodimer mediates derepression.

    PubMed

    Terán, Wilson; Krell, Tino; Ramos, Juan Luis; Gallegos, María-Trinidad

    2006-03-17

    The RND family transporter TtgABC and its cognate repressor TtgR from Pseudomonas putida DOT-T1E were both shown to possess multidrug recognition properties. Structurally unrelated molecules such as chloramphenicol, butyl paraben, 1,3-dihydroxynaphthalene, and several flavonoids are substrates of TtgABC and activate pump expression by binding to the TtgR-operator complex. Isothermal titration calorimetry was employed to determine the thermodynamic parameters for the binding of these molecules to TtgR. Dissociation constants were in the range from 1 to 150 microm, the binding stoichiometry was one effector molecule per dimer of TtgR, and the process was driven by favorable enthalpy changes. Although TtgR exhibits a large multidrug binding profile, the plant-derived compounds phloretin and quercetin were shown to bind with the highest affinity (K(D) of around 1 microm), in contrast to other effectors (chloramphenicol and aromatic solvents) for which exhibited a more reduced affinity. Structure-function studies of effectors indicate that the presence of aromatic rings as well as hydroxyl groups are determinants for TtgR binding. The binding of TtgR to its operator DNA does not alter the protein effector profile nor the effector binding stoichiometry. Moreover, we demonstrate here for the first time that the binding of a single effector molecule to the DNA-bound TtgR homodimer induces the dissociation of the repressor-operator complex. This provides important insight into the molecular mechanism of effector-mediated derepression.

  6. Comprehensive analysis of animal TALE homeobox genes: new conserved motifs and cases of accelerated evolution.

    PubMed

    Mukherjee, Krishanu; Bürglin, Thomas R

    2007-08-01

    TALE homeodomain proteins are an ancient subgroup within the group of homeodomain transcription factors that play important roles in animal, plant, and fungal development. We have extracted the full complement of TALE superclass homeobox genes from the genome projects of seven protostomes, seven deuterostomes, and Nematostella. This was supplemented with TALE homeobox genes from additional species and phylogenetic analyses were carried out with 276 sequences. We found 20 homeobox genes and 4 pseudogenes in humans, 21 genes in mouse, 8 genes in Drosophila, and 5 genes plus one truncated gene in Caenorhabditis elegans. Apart from the previously identified TALE classes MEIS, PBC, IRO, and TGIF, a novel class is identified, termed MOHAWK (MKX). Further, we show that the MEIS class can be divided into two families, PREP and MEIS. Prep genes have previously only been described in vertebrates but are lacking in Drosophila. Here we identify orthologues in other insect taxa as well as in the cnidarian Nematostella. In C. elegans, a divergent Prep protein has lost the homeodomain. Full-length multiple sequence alignment of the protostome and deuterostome sequences allowed us to identify several novel conserved motifs within the MKX, TGIF, and MEIS classes. Phylogenetic analyses revealed fast-evolving PBC class genes; in particular, some X-linked PBC genes in nematodes are subject to rapid evolution. In addition, several instances of gene loss were identified. In conclusion, our comprehensive analysis provides a defining framework for the classification of animal TALE homeobox genes and the understanding of their evolution.

  7. Type III secretion chaperones of Pseudomonas syringae protect effectors from Lon-associated degradation.

    PubMed

    Losada, Liliana C; Hutcheson, Steven W

    2005-02-01

    The hrp type III secretion system (TTSS) of Pseudomonas syringae translocates effector proteins into the cytoplasm of host cells. Proteolysis of HrpR by Lon has been shown to negatively regulate the hrp TTSS. The inability to bypass Lon-associated effects on the regulatory system by ectopic expression of the known regulators suggested a second site of action for Lon in TTSS-dependent effector secretion. In this study we report that TTSS-dependent effectors are subject to the proteolytic degradation that appears to be rate-limiting to secretion. The half-lives of the effectors AvrPto, AvrRpt2, HopPsyA, HopPsyB1, HopPtoB2, HopPsyV1, HopPtoG and HopPtoM were substantially higher in bacteria lacking Lon. TTSS-dependent secretion of several effectors was enhanced from Lon mutants. A primary role for chaperones appears to be protection of effectors from Lon-associated degradation prior to secretion. When coexpressed with their cognate chaperone, HopPsyB1, HopPsyV1 and HopPtoM were at least 10 times more stable in strains expressing Lon. Distinct Lon-targeting and chaperone-binding domains were identified in HopPtoM. The results imply that Lon is involved at two distinct levels in the regulation of the P. syringae TTSS: regulation of assembly of the secreton and modulation of effector secretion.

  8. Blockade of TNF-α signaling benefits cancer therapy by suppressing effector regulatory T cell expansion.

    PubMed

    Chang, Li-Yuan; Lin, Yung-Chang; Chiang, Jy-Ming; Mahalingam, Jayashri; Su, Shih-Huan; Huang, Ching-Tai; Chen, Wei-Ting; Huang, Chien-Hao; Jeng, Wen-Juei; Chen, Yi-Cheng; Lin, Shi-Ming; Sheen, I-Shyan; Lin, Chun-Yen

    2015-10-01

    Effector but not naive regulatory T cells (Treg cells) can accumulate in the peripheral blood as well as the tumor microenvironment, expand during tumor progression and be one of the main suppressors for antitumor immunity. However, the underlying mechanisms for effector Treg cell expansion in tumor are still unknown. We demonstrate that effector Treg cell-mediated suppression of antitumor CD8(+) T cells is tumor-nonspecific. Furthermore, TNFR2 expression is increased in these Treg cells by Affymetrix chip analysis which was confirmed by monoclonal antibody staining in both hepatocellular carcinoma (HCC) and colorectal cancer (CRC) patients and murine models. Correspondingly, increased levels of TNF-α in both tissue and serum were also demonstrated. Interestingly, TNF-α could not only expand effector Treg cells through TNFR2 signaling, but also enhanced their suppressive activity against antitumor immunity of CD8(+) T cells. Furthermore, targeting TNFR2 signaling with a TNF-α inhibitor could selectively reduce rapid resurgence of effector Treg cells after cyclophosphamide-induced lymphodepletion and markedly inhibit the growth of established tumors. Herein, we propose a novel mechanism in which TNF-α could promote tumor-associated effector Treg cell expansion and suggest a new cancer immunotherapy strategy using TNF-α inhibitors to reduce effector Treg cells expansion after cyclophosphamide-induced lymphodepletion.

  9. Manipulation of plant cells by cyst and root-knot nematode effectors.

    PubMed

    Hewezi, Tarek; Baum, Thomas J

    2013-01-01

    A key feature of sedentary plant-parasitic nematodes is the release of effector proteins from their esophageal gland cells through their stylets into host roots. These proteinaceous stylet secretions have been shown to be crucial for successful parasitism by mediating the transition of normal root cells into specialized feeding sites and by negating plant defenses. Recent technical advances of purifying mRNA from esophageal gland cells of plant-parasitic nematodes coupled with emerging sequencing technologies is steadily expanding our knowledge of nematode effector repertoires. Host targets and biological activities of a number of nematode effectors are continuously being reported and, by now, a first picture of the complexity of sedentary nematode parasitism at the molecular level is starting to take shape. In this review, we highlight effector mechanisms that recently have been uncovered by studying the host-pathogen interaction. These mechanisms range from mediating susceptibility of host plants to the actual triggering of defense responses. In particular, we portray and discuss the mechanisms by which nematode effectors modify plant cell walls, negate host defense responses, alter auxin and polyamine signaling, mimic plant molecules, regulate stress signaling, and activate hypersensitive responses. Continuous molecular characterization of newly discovered nematode effectors will be needed to determine how these effectors orchestrate host signaling pathways and biological processes leading to successful parasitism.

  10. Dendritic cell-derived nitric oxide inhibits the differentiation of effector dendritic cells

    PubMed Central

    Wu, Tianshu; Lu, Geming; Hu, Yuan; Zhang, Hui; Xu, Feihong; Wei, Peter; Chen, Kang; Tang, Hua; Yeretssian, Garabet; Xiong, Huabao

    2016-01-01

    Dendritic cells (DCs) play a pivotal role in the development of effective immune defense while avoiding detrimental inflammation and autoimmunity by regulating the balance of adaptive immunity and immune tolerance. However, the mechanisms that govern the effector and regulatory functions of DCs are incompletely understood. Here, we show that DC-derived nitric oxide (NO) controls the balance of effector and regulatory DC differentiation. Mice deficient in the NO-producing enzyme inducible nitric oxide synthase (iNOS) harbored increased effector DCs that produced interleukin-12, tumor necrosis factor (TNF) and IL-6 but normal numbers of regulatory DCs that expressed IL-10 and programmed cell death-1 (PD-1). Furthermore, an iNOS-specific inhibitor selectively enhanced effector DC differentiation, mimicking the effect of iNOS deficiency in mice. Conversely, an NO donor significantly suppressed effector DC development. Furthermore, iNOS−/− DCs supported enhanced T cell activation and proliferation. Finally iNOS−/− mice infected with the enteric pathogen Citrobacter rodentium suffered more severe intestinal inflammation with concomitant expansion of effector DCs in colon and spleen. Collectively, our results demonstrate that DC-derived iNOS restrains effector DC development, and offer the basis of therapeutic targeting of iNOS in DCs to treat autoimmune and inflammatory diseases. PMID:27556858

  11. Entry of oomycete and fungal effectors into plant and animal host cells.

    PubMed

    Kale, Shiv D; Tyler, Brett M

    2011-12-01

    Fungal and oomycete pathogens cause many destructive diseases of plants and important diseases of humans and other animals. Fungal and oomycete plant pathogens secrete numerous effector proteins that can enter inside host cells to condition susceptibility. Until recently it has been unknown if these effectors enter via pathogen-encoded translocons or via pathogen-independent mechanisms. Here we review recent evidence that many fungal and oomycete effectors enter via receptor-mediated endocytosis, and can do so in the absence of the pathogen. Surprisingly, a large number of these effectors utilize cell surface phosphatidyinositol-3-phosphate (PI-3-P) as a receptor, a molecule previously known only inside cells. Binding of effectors to PI-3-P appears to be mediated by the cell entry motif RXLR in oomycetes, and by diverse RXLR-like variants in fungi. PI-3-P appears to be present on the surface of animal cells also, suggesting that it may mediate entry of effectors of fungal and oomycete animal pathogens, for example, RXLR effectors found in the oomycete fish pathogen, Saprolegnia parasitica. Reagents that can block PI-3-P-mediated entry have been identified, suggesting new therapeutic strategies.

  12. Studying the Mechanism of Plasmopara viticola RxLR Effectors on Suppressing Plant Immunity

    PubMed Central

    Xiang, Jiang; Li, Xinlong; Wu, Jiao; Yin, Ling; Zhang, Yali; Lu, Jiang

    2016-01-01

    The RxLR effector family, produced by oomycete pathogens, may manipulate host physiological and biochemical events inside host cells. A group of putative RxLR effectors from Plasmopara viticola have been recently identified by RNA-Seq analysis in our lab. However, their roles in pathogenesis are poorly understood. In this study, we attempted to characterize 23 PvRxLR effector candidates identified from a P. viticola isolate “ZJ-1-1.” During host infection stages, expression patterns of the effector genes were varied and could be categorized into four different groups. By using transient expression assays in Nicotiana benthamiana, we found that 17 of these effector candidates fully suppressed programmed cell death elicited by a range of cell death-inducing proteins, including BAX, INF1, PsCRN63, PsojNIP, PvRxLR16 and R3a/Avr3a. We also discovered that all these PvRxLRs could target the plant cell nucleus, except for PvRxLR55 that localized to the membrane. Furthermore, we identified a single effector, PvRxLR28, that showed the highest expression level at 6 hpi. Functional analysis revealed that PvRxLR28 could significantly enhance susceptibilities of grapevine and tobacco to pathogens. These results suggest that most P. viticola effectors tested in this study may act as broad suppressors of cell death to manipulate immunity in plant. PMID:27242731

  13. Bacterial effector HopF2 interacts with AvrPto and suppresses Arabidopsis innate immunity at the plasma membrane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant pathogenic bacteria inject a cocktail of effector proteins into host plant cells to modulate the host immune response, thereby promoting pathogenicity. How or whether these effectors work cooperatively is largely unknown. The Pseudomonas syringae DC3000 effector HopF2 suppresses the host plan...

  14. Strain Specific Factors Control Effector Gene Silencing in Phytophthora sojae

    PubMed Central

    Shrestha, Sirjana Devi; Chapman, Patrick; Zhang, Yun; Gijzen, Mark

    2016-01-01

    The Phytophthora sojae avirulence gene Avr3a encodes an effector that is capable of triggering immunity on soybean plants carrying the resistance gene Rps3a. P. sojae strains that express Avr3a are avirulent to Rps3a plants, while strains that do not are virulent. To study the inheritance of Avr3a expression and virulence towards Rps3a, genetic crosses and self-fertilizations were performed. A cross between P. sojae strains ACR10 X P7076 causes transgenerational gene silencing of Avr3a allele, and this effect is meiotically stable up to the F5 generation. However, test-crosses of F1 progeny (ACR10 X P7076) with strain P6497 result in the release of silencing of Avr3a. Expression of Avr3a in the progeny is variable and correlates with the phenotypic penetrance of the avirulence trait. The F1 progeny from a direct cross of P6497 X ACR10 segregate for inheritance for Avr3a expression, a result that could not be explained by parental imprinting or heterozygosity. Analysis of small RNA arising from the Avr3a gene sequence in the parental strains and hybrid progeny suggests that the presence of small RNA is necessary but not sufficient for gene silencing. Overall, we conclude that inheritance of the Avr3a gene silenced phenotype relies on factors that are variable among P. sojae strains. PMID:26930612

  15. Crystal Structure of the Salmonella Typhimurium Effector GtgE

    PubMed Central

    Wong, Kathy; Gehring, Kalle; Cygler, Miroslaw

    2016-01-01

    Salmonella Typhimurium GtgE is an effector protein contributing to the virulence of this pathogen. It was shown to possess highly selective proteolytic activity against a subset of Rab proteins that helps in evasion of Salmonella-containing vacuole (SCV) fusion with lysosomes. Cys45, His151 and Asp169 are essential for proteolytic activity. The structure of a C-terminal fragment GtgE(79–214) indicated the presence of a papain-like fold. Here, we present the structure of GtgE(17–214) containing the fully assembled active site. The design of a proteolytically active and crystallizable GtgE construct was aided by NMR spectroscopy. The protein indeed displays papain-like fold with an assembled Cys-His-Asp catalytic triad. Like the full-length GtgE, the crystallizable construct showed low activity in vitro for its known substrates, Rab32 and Rab29. NMR titration experiments showed at most very weak binding of GtgE to the peptide encompassing the Rab29 cleavage site. In view of the low in vitro activity and poor substrate binding, we postulate that the function of GtgE in vivo as a proteolytic enzyme is dependent on other factor(s), such as a protein partner or interactions with the SCV membrane, which stimulate(s) GtgE activity in vivo. PMID:27923041

  16. Rubicon controls endosome maturation as a Rab7 effector.

    PubMed

    Sun, Qiming; Westphal, Wiebke; Wong, Kwun Ngok; Tan, Irena; Zhong, Qing

    2010-11-09

    The activation and recruitment of the small GTPase Rab7 to early endosome is a critical step for early to late endosome maturation, a process that requires the class III phosphatidylinositol 3-kinase (PI3KC3) and GTPase regulators. However, the molecular mechanism underlying Rab7 activation and endosome maturation is still poorly defined. Here we report that Rubicon, a component of the PI3KC3 complex, prevents endosome maturation through differential interactions with Rab7 and UVRAG. UVRAG activates PI3KC3 and C-VPS/HOPS, a guanine nucleotide exchange factor that catalyzes the exchange of GDP for GTP on Rab7. We demonstrate that Rubicon sequesters UVRAG from C-VPS/HOPS. Active GTP-bound Rab7 competes for Rubicon binding and releases UVRAG to associate with C-VPS/HOPS, which in turn promotes further loading of Rab7 with GTP. This feed-forward loop ensures rapid amplification of GTP-bound Rab7 and consequent stimulation of endosome maturation. Hence, Rubicon serves as a previously unknown Rab7 effector to ensure the proper progression of the endocytic pathway.

  17. The molecular makeup and function of regulatory and effector synapses.

    PubMed

    Reichardt, Peter; Dornbach, Bastian; Gunzer, Matthias

    2007-08-01

    Physical interactions between T cells and antigen-presenting cells (APCs) form the basis of any specific immune response. Upon cognate contacts, a multimolecular assembly of receptors and adhesion molecules on both cells is created, termed the immunological synapse (IS). Very diverse structures of ISs have been described, yet the functional importance for T-cell differentiation is largely unclear. Here we discuss the principal structure and function of ISs. We then focus on two characteristic T-cell-APC pairs, namely T cells contacting dendritic cells (DCs) or naive B cells, for which extremely different patterns of the IS have been observed as well as fundamentally different effects on the function of the activated T cells. We provide a model on how differences in signaling and the involvement of adhesion molecules might lead to diverse interaction kinetics and, eventually, diverse T-cell differentiation. We hypothesize that the preferred activation of the adhesion molecule leukocyte function-associated antigen-1 (LFA-1) and of the negative regulator for T-cell activation, cytotoxic T-lymphocyte antigen-4 (CTLA-4), through contact with naive B cells, lead to prolonged cell-cell contacts and the generation of T cells with regulatory capacity. In contrast, DCs might have evolved mechanisms to avoid LFA-1 overactivation and CTLA-4 triggering, thereby promoting more dynamic contacts that lead to the preferential generation of effector cells.

  18. Evaluation of Nod-Like Receptor (NLR) Effector Domain Interactions

    PubMed Central

    Kufer, Thomas A.; Schwarzenbacher, Robert

    2009-01-01

    Members of the Nod-like receptor (NLR) family recognize intracellular pathogens and recruit a variety of effector molecules, including pro-caspases and kinases, which in turn are implicated in cytokine processing and NF-κB activation. In order to elucidate the intricate network of NLR signaling, which is still fragmentary in molecular terms, we applied comprehensive yeast two-hybrid analysis for unbiased evaluation of physical interactions between NLRs and their adaptors (ASC, CARD8) as well as kinase RIPK2 and inflammatory caspases (C1, C2, C4, C5) under identical conditions. Our results confirmed the interaction of NOD1 and NOD2 with RIPK2, and between NLRP3 and ASC, but most importantly, our studies revealed hitherto unrecognized interactions of NOD2 with members of the NLRP subfamily. We found that NOD2 specifically and directly interacts with NLRP1, NLRP3 and NLRP12. Furthermore, we observed homodimerization of the RIPK2 CARD domains and identified residues in NOD2 critical for interaction with RIPK2. In conclusion, our work provides further evidence for the complex network of protein-protein interactions underlying NLR function. PMID:19337385

  19. Plasma Aerodynamic Control Effectors for Improved Wind Turbine Performance

    SciTech Connect

    Mehul P. Patel; Srikanth Vasudevan; Robert C. Nelson; Thomas C. Corke

    2008-08-01

    Orbital Research Inc is developing an innovative Plasma Aerodynamic Control Effectors (PACE) technology for improved performance of wind turbines. The PACE system is aimed towards the design of "smart" rotor blades to enhance energy capture and reduce aerodynamic loading and noise using flow-control. The PACE system will provide ability to change aerodynamic loads and pitch distribution across the wind turbine blade without any moving surfaces. Additional benefits of the PACE system include reduced blade structure weight and complexity that should translate into a substantially reduced initial cost. During the Phase I program, the ORI-UND Team demonstrated (proof-of-concept) performance improvements on select rotor blade designs using PACE concepts. Control of both 2-D and 3-D flows were demonstrated. An analytical study was conducted to estimate control requirements for the PACE system to maintain control during wind gusts. Finally, independent laboratory experiments were conducted to identify promising dielectric materials for the plasma actuator, and to examine environmental effects (water and dust) on the plasma actuator operation. The proposed PACE system will be capable of capturing additional energy, and reducing aerodynamic loading and noise on wind turbines. Supplementary benefits from the PACE system include reduced blade structure weight and complexity that translates into reduced initial capital costs.

  20. The RAS-Effector Interaction as a Drug Target.

    PubMed

    Keeton, Adam B; Salter, E Alan; Piazza, Gary A

    2017-01-15

    About a third of all human cancers harbor mutations in one of the K-, N-, or HRAS genes that encode an abnormal RAS protein locked in a constitutively activated state to drive malignant transformation and tumor growth. Despite more than three decades of intensive research aimed at the discovery of RAS-directed therapeutics, there are no FDA-approved drugs that are broadly effective against RAS-driven cancers. Although RAS proteins are often said to be "undruggable," there is mounting evidence suggesting it may be feasible to develop direct inhibitors of RAS proteins. Here, we review this evidence with a focus on compounds capable of inhibiting the interaction of RAS proteins with their effectors that transduce the signals of RAS and that drive and sustain malignant transformation and tumor growth. These reports of direct-acting RAS inhibitors provide valuable insight for further discovery and development of clinical candidates for RAS-driven cancers involving mutations in RAS genes or otherwise activated RAS proteins. Cancer Res; 77(2); 221-6. ©2017 AACR.

  1. Receptor-coupled effector systems and their interactions

    SciTech Connect

    Wiener, E.C.

    1988-01-01

    We investigated the modulation of intracellular signal generation by receptor-coupled effector systems in B lymphocytes, and whether these alterations are consistent with the effects of prostaglandins. TPA (12-O-tetradecanoyl phorbol-13-acetate) and sn-1,2,-dioctanoylglycerol (diC{sub 8}) substitute for lipid derived signals which activate protein kinase C. Pretreating splenocytes from athymic nude mice with 100nM TPA or 5 {mu}M diC{sub 8} potentiated the forskolin-induced increased in cAMP (measured by radioimmunoassay) 2.5 and 3.0 times (respectively), but they decreased the PGE{sub 1}-induced cAMP rise 48% and 35% (respectively). Goat anti-mouse IgM, which activates diacylglycerol production, potentiated the forskolin-induced cAMP increase by 76%, but reduced that of PGE{sub 1} by 30%. Rabbit anti-mouse IgG, its F(ab{prime}){sub 2} fragment, or goat anti-mouse IGM induced increases in the cytosolic free (Ca{sup 2+}), (Ca{sup 2+}){sub i}, which TPA inhibited. In contrast, TPA potential antibody-induced {sup 3}H-thymidine (85x) and {sup 3}H-uridine (30x) uptake in B lymphocytes.

  2. Exosomes: novel effectors of human platelet lysate activity.

    PubMed

    Torreggiani, E; Perut, F; Roncuzzi, L; Zini, N; Baglìo, S R; Baldini, N

    2014-09-22

    Despite the popularity of platelet-rich plasma (PRP) and platelet lysate (PL) in orthopaedic practice, the mechanism of action and the effectiveness of these therapeutic tools are still controversial. So far, the activity of PRP and PL has been associated with different growth factors (GF) released during platelet degranulation. This study, for the first time, identifies exosomes, nanosized vesicles released in the extracellular compartment by a number of elements, including platelets, as one of the effectors of PL activity. Exosomes were isolated from human PL by differential ultracentrifugation, and analysed by electron microscopy and Western blotting. Bone marrow stromal cells (MSC) treated with three different exosome concentrations (0.6 μg, 5 μg and 50 μg) showed a significant, dose-dependent increase in cell proliferation and migration compared to the control. In addition, osteogenic differentiation assays demonstrated that exosome concentration differently affected the ability of MSC to deposit mineralised matrix. Finally, the analysis of exosome protein content revealed a higher amount of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) as compared to PL. In regards to RNA content, an enrichment of small RNAs in exosomes as compared to donor platelets has been found. These results suggest that exosomes consistently contribute to PL activity and could represent an advantageous nanodelivery system for cell-free regeneration therapies.

  3. Suppression of HIV-1 replication by microRNA effectors

    PubMed Central

    Chable-Bessia, Christine; Meziane, Oussama; Latreille, Daniel; Triboulet, Robinson; Zamborlini, Alessia; Wagschal, Alexandre; Jacquet, Jean-Marc; Reynes, Jacques; Levy, Yves; Saib, Ali; Bennasser, Yamina; Benkirane, Monsef

    2009-01-01

    The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART. PMID:19272132

  4. Autoimmune effector memory T cells: the bad and the good

    PubMed Central

    Devarajan, Priyadharshini; Chen, Zhibin

    2014-01-01

    Immunological memory is a hallmark of adaptive immunity, a defense mechanism endowed to vertebrates during evolution. However, an autoimmune pathogenic role of memory lymphocytes is also emerging with accumulating evidence, despite reasonable skepticism on their existence in a chronic setting of autoimmune damage. It is conceivable that autoimmune memory would be particularly harmful since memory cells would constantly “remember” and attack the body's healthy tissues. It is even more detrimental given the resistance of memory T cells to immunomodulatory therapies. In this review, we focus on self-antigen-reactive CD4+ effector memory T (TEM) cells, surveying the evidence for the role of the TEM compartment in autoimmune pathogenesis. We will also discuss the role of TEM cells in chronic and acute infectious disease settings and how they compare to their counterparts in autoimmune diseases. With their long-lasting potency, the autoimmune TEM cells could also play a critical role in anti-tumor immunity, which may be largely based on their reactivity to self-antigens. Therefore, although autoimmune TEM cells are “bad” due to their role in relentless perpetration of tissue damage in autoimmune disease settings, they are unlikely a by-product of industrial development along the modern surge of autoimmune disease prevalence. Rather, they may be a product of evolution for their “good” in clearing damaged host cells in chronic infections and malignant cells in cancer settings. PMID:24203440

  5. A fan tale, modern and ancient fans - A comparison

    SciTech Connect

    Fischer, P.J. ); Thor, D.R. ); Cherven, V.B.

    1991-02-01

    The Quaternary Conception fan of the Santa Barbara basin and the Upper Cretaceous Lathrop fan of the northern San Joaquin basin tell an interesting tale. Both fans show a well defined sequence stratigraphy of alternating low-stand, sand-rich units that alternate with thin high-stand silt units that drape and in-fill the surface topography of the previous sand-cycle. Isopachs made from detailed well log correlations (Lathrop) and seismic reflection data tied to borings (Conception) show that the fans are composed of a series of offset-stacked, elongate fan lobes. These lobes are similar in size. A major difference in the development of the two fans is the timing of tectonism. Concomitant tectonism uplifted the Conception fan lobes and resulted in localized erosion of high-stand silts beds and sand-on-sand lobe contacts. Tectonism and Lathrop occurred after fan deposition and provided the trapping structure-the Lathrop anticlinal fold. Following are some lessons to be learned from these and other fans the authors have studied: (1) Quaternary or modern' fans and ancient fans are similar. (2) Elongate sand-rich fan lobes separated by highstand silt units are typical of fans. (3) In addition to well-known techniques (seismic stratigraphy and detailed well log correlations), original reservoir pressures may be used to differentiate sequences and lobes (e.g., Lathrop). (4) Tectonism and erosion along the margin may limit traps to the uppermost lobe sequence (e.g., Conception). (5) An offset-stacked elongate fan lobe model is a valuable exploration and production tool.

  6. The Cell Death Triggered by the Nuclear Localized RxLR Effector PITG_22798 from Phytophthora infestans Is Suppressed by the Effector AVR3b.

    PubMed

    Wang, Hongyang; Ren, Yajuan; Zhou, Jing; Du, Juan; Hou, Juan; Jiang, Rui; Wang, Haixia; Tian, Zhendong; Xie, Conghua

    2017-02-14

    Phytopathogenic oomycetes, such as Phytophthora infestans, potentially secrete many RxLR effector proteins into plant cells to modulate plant immune responses and promote colonization. However, the molecular mechanisms by which these RxLR effectors suppress plant immune responses are largely unknown. Here we describe an RxLR effector PITG_22798 (Gene accession: XM_002998349) that was upregulated during early infection of potato by P. infestans. By employment of agroinfiltration, we observed that PITG_22798 triggers cell death in Nicotiana benthamiana. Confocal microscopic examination showed that PITG_22798-GFP (Green Fluorescent Protein) located in the host nucleus when expressed transiently in N. benthamiana leaves. A nuclear localization signal (NLS) domain of PITG_22798 is important for nuclear localization and cell death-inducing activity. Sequence alignment and transient expression showed that PITG_22798 from diverse P. infestans isolates are conserved, and transient expression of PITG_22798 enhances P. infestans colonization of N. benthamiana leaves, which suggests that PITG_22798 contributes to P. infestans infection. PITG_22798-triggered cell death is dependent on SGT1-mediated signaling and is suppressed by the P. infestans avirulence effector 3b (AVR3b). The present research provides a clue for further investigation of how P. infestans effector PITG_22798 associates with and modulates host immunity.

  7. The Cell Death Triggered by the Nuclear Localized RxLR Effector PITG_22798 from Phytophthora infestans Is Suppressed by the Effector AVR3b

    PubMed Central

    Wang, Hongyang; Ren, Yajuan; Zhou, Jing; Du, Juan; Hou, Juan; Jiang, Rui; Wang, Haixia; Tian, Zhendong; Xie, Conghua

    2017-01-01

    Phytopathogenic oomycetes, such as Phytophthora infestans, potentially secrete many RxLR effector proteins into plant cells to modulate plant immune responses and promote colonization. However, the molecular mechanisms by which these RxLR effectors suppress plant immune responses are largely unknown. Here we describe an RxLR effector PITG_22798 (Gene accession: XM_002998349) that was upregulated during early infection of potato by P. infestans. By employment of agroinfiltration, we observed that PITG_22798 triggers cell death in Nicotiana benthamiana. Confocal microscopic examination showed that PITG_22798-GFP (Green Fluorescent Protein) located in the host nucleus when expressed transiently in N. benthamiana leaves. A nuclear localization signal (NLS) domain of PITG_22798 is important for nuclear localization and cell death-inducing activity. Sequence alignment and transient expression showed that PITG_22798 from diverse P. infestans isolates are conserved, and transient expression of PITG_22798 enhances P. infestans colonization of N. benthamiana leaves, which suggests that PITG_22798 contributes to P. infestans infection. PITG_22798-triggered cell death is dependent on SGT1-mediated signaling and is suppressed by the P. infestans avirulence effector 3b (AVR3b). The present research provides a clue for further investigation of how P. infestans effector PITG_22798 associates with and modulates host immunity. PMID:28216607

  8. Motor resonance in left- and right-handers: evidence for effector-independent motor representations

    PubMed Central

    Sartori, Luisa; Begliomini, Chiara; Castiello, Umberto

    2012-01-01

    The idea of motor resonance was born at the time that it was demonstrated that cortical and spinal pathways of the motor system are specifically activated during both action-observation and execution. What is not known is if the human action observation-execution matching system simulates actions through motor representations specifically attuned to the laterality of the observed effectors (i.e., effector-dependent representations) or through abstract motor representations unconnected to the observed effector (i.e., effector-independent representations). To answer that question we need to know how the information necessary for motor resonance is represented or integrated within the representation of an effector. Transcranial magnetic stimulation (TMS)-induced motor evoked potentials (MEPs) were thus recorded from the dominant and non-dominant hands of left- and right-handed participants while they observed a left- or a right-handed model grasping an object. The anatomical correspondence between the effector being observed and the observer's effector classically reported in the literature was confirmed by the MEP response in the dominant hand of participants observing models with their same hand preference. This effect was found in both left- as well as in right-handers. When a broader spectrum of options, such as actions performed by a model with a different hand preference, was instead considered, that correspondence disappeared. Motor resonance was noted in the observer's dominant effector regardless of the laterality of the hand being observed. This would indicate that there is a more sophisticated mechanism which works to convert someone else's pattern of movement into the observer's optimal motor commands and that effector-independent representations specifically modulate motor resonance. PMID:23408666

  9. Space-based multifunctional end effector systems functional requirements and proposed designs

    NASA Technical Reports Server (NTRS)

    Mishkin, A. H.; Jau, B. M.

    1988-01-01

    The end effector is an essential element of teleoperator and telerobot systems to be employed in space in the next decade. The report defines functional requirements for end effector systems to perform operations that are currently only feasible through Extra-Vehicular Activity (EVA). Specific tasks and functions that the end effectors must be capable of performing are delineated. Required capabilities for forces and torques, clearances, compliance, and sensing are described, using current EVA requirements as guidelines where feasible. The implications of these functional requirements on the elements of potential end effector systems are discussed. The systems issues that must be considered in the design of space-based manipulator systems are identified; including impacts on subsystems tightly coupled to the end effector, i.e., control station, information processing, manipulator arm, tool and equipment stowage. Possible end effector designs are divided into three categories: single degree-of-freedom end effectors, multiple degree of freedom end effectors, and anthropomorphic hands. Specific design alternatives are suggested and analyzed within the individual categories. Two evaluations are performed: the first considers how well the individual end effectors could substitute for EVA; the second compares how manipulator systems composed of the top performers from the first evaluation would improve the space shuttle Remote Manipulator System (RMS) capabilities. The analysis concludes that the anthropomorphic hand is best-suited for EVA tasks. A left- and right-handed anthropomorphic manipulator arm configuration is suggested as appropriate to be affixed to the RMS, but could also be used as part of the Smart Front End for the Orbital Maneuvering Vehicle (OMV). The technical feasibility of the anthropomorphic hand and its control are demonstrated. An evolutionary development approach is proposed and approximate scheduling provided for implementing the suggested

  10. Requirements for Driving Antipathogen Effector Genes into Populations of Disease Vectors by Homing

    PubMed Central

    Beaghton, Andrea; Hammond, Andrew; Nolan, Tony; Crisanti, Andrea; Godfray, H. Charles J.; Burt, Austin

    2017-01-01

    There is a need for new interventions against the ongoing burden of vector-borne diseases such as malaria and dengue. One suggestion has been to develop genes encoding effector molecules that block parasite development within the vector, and then use the nuclease-based homing reaction as a form of gene drive to spread those genes through target populations. If the effector gene reduces the fitness of the mosquito and does not contribute to the drive, then loss-of-function mutations in the effector will eventually replace functional copies, but protection may nonetheless persist sufficiently long to provide a public health benefit. Here, we present a quantitative model allowing one to predict the duration of protection as a function of the probabilities of different molecular processes during the homing reaction, various fitness effects, and the efficacy of the effector in blocking transmission. Factors that increase the duration of protection include reducing the frequency of pre-existing resistant alleles, the probability of nonrecombinational DNA repair, the probability of homing-associated loss of the effector, the fitness costs of the nuclease and effector, and the completeness of parasite blocking. For target species that extend over an area much larger than the typical dispersal distance, the duration of protection is expected to be highest at the release site, and decrease away from there, eventually falling to zero, as effector-less drive constructs replace effector-containing ones. We also model an alternative strategy of using the nuclease to target an essential gene, and then linking the effector to a sequence that restores the essential function and is resistant to the nuclease. Depending upon parameter values, this approach can prolong the duration of protection. Our models highlight the key design criteria needed to achieve a desired level of public health benefit. PMID:28159753

  11. Glimpse into Hox and tale regulation of cell differentiation and reprogramming.

    PubMed

    Cerdá-Esteban, Nuria; Spagnoli, Francesca M

    2014-01-01

    During embryonic development, cells become gradually restricted in their developmental potential and start elaborating lineage-specific transcriptional networks to ultimately acquire a unique differentiated state. Hox genes play a central role in specifying regional identities, thereby providing the cell with critical information on positional value along its differentiation path. The exquisite DNA-binding specificity of the Hox proteins is frequently dependent upon their interaction with members of the TALE family of homeodomain proteins. In addition to their function as Hox-cofactors, TALE homeoproteins control multiple crucial developmental processes through Hox-independent mechanisms. Here, we will review recent findings on the function of both Hox and TALE proteins in cell differentiation, referring mostly to vertebrate species. In addition, we will discuss the direct implications of this knowledge on cell plasticity and cell reprogramming.

  12. Targeted repression of AXIN2 and MYC gene expression using designer TALEs

    SciTech Connect

    Rennoll, Sherri A.; Scott, Samantha A.; Yochum, Gregory S.

    2014-04-18

    Highlights: • We designed TALE–SID fusion proteins to target AXIN2 and MYC. • TALE–SIDs bound the chromosomal AXIN2 and MYC genes and repressed their expression. • TALE–SIDs repress β-catenin{sup S45F}-dependent AXIN2 and MYC transcription. - Abstract: Designer TALEs (dTALEs) are chimeric transcription factors that can be engineered to regulate gene expression in mammalian cells. Whether dTALEs can block gene transcription downstream of signal transduction cascades, however, has yet to be fully explored. Here we tested whether dTALEs can be used to target genes whose expression is controlled by Wnt/β-catenin signaling. TALE DNA binding domains were engineered to recognize sequences adjacent to Wnt responsive enhancer elements (WREs) that control expression of axis inhibition protein 2 (AXIN2) and c-MYC (MYC). These custom DNA binding domains were linked to the mSin3A interaction domain (SID) to generate TALE–SID chimeric repressors. The TALE–SIDs repressed luciferase reporter activity, bound their genomic target sites, and repressed AXIN2 and MYC expression in HEK293 cells. We generated a novel HEK293 cell line to determine whether the TALE–SIDs could function downstream of oncogenic Wnt/β-catenin signaling. Treating these cells with doxycycline and tamoxifen stimulates nuclear accumulation of a stabilized form of β-catenin found in a subset of colorectal cancers. The TALE–SIDs repressed AXIN2 and MYC expression in these cells, which suggests that dTALEs could offer an effective therapeutic strategy for the treatment of colorectal cancer.

  13. Alte Marchen?--alter Hut. Neue Marchen!--neuer Hut? 3 Moderne Marchen bearbeitet als Horspiele, mit Aufgaben und Ubungen (Old Fairy Tales?--Old Hat. New Fairy Tales!--New Hat? Three Modern Fairy Tales Presented in a Radio Play, with Activities and Drills).

    ERIC Educational Resources Information Center

    Kage, Michael

    Three German modern "fairy tales" are presented in a radio play format with exercises based on the tales. Detailed suggestions for the exercises and other class activities based on the manual are included. While serving as a comprehension exercise of the preceding text, the exercises and other activities recommended to accompany them depart from…

  14. Psychoanalysis and detective fiction: a tale of Freud and criminal storytelling.

    PubMed

    Yang, Amy

    2010-01-01

    Much has been written about Freud's influence on popular culture. This article addresses the influence of literature on Freud's psychoanalytical theory, specifically the role that modern detective fiction played in shaping Freudian theory. Edgar Allan Poe gave Freud the literary precedent; Sir Arthur Conan Doyle's creation Sherlock Holmes gave him the analytical model. In turn, the world of crime story-telling embedded Freudian theories in subsequent forms, spinning the tales of crime into a journey into the human mind. As these tales were popularized on the silver screen in the early 20th century, psychoanalytical ideas moved from the lecture halls into the cultural mainstream.

  15. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain.

    PubMed

    Li, Ting; Huang, Sheng; Jiang, Wen Zhi; Wright, David; Spalding, Martin H; Weeks, Donald P; Yang, Bing

    2011-01-01

    DNA double-strand breaks enhance homologous recombination in cells and have been exploited for targeted genome editing through use of engineered endonucleases. Here we report the creation and initial characterization of a group of rare-cutting, site-specific DNA nucleases produced by fusion of the restriction enzyme FokI endonuclease domain (FN) with the high-specificity DNA-binding domains of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are members of the transcription activator-like (TAL) effector family whose central repeat units dictate target DNA recognition and can be modularly constructed to create novel DNA specificity. The hybrid FN-AvrXa7, AvrXa7-FN and PthXo1-FN proteins retain both recognition specificity for their target DNA (a 26 bp sequence for AvrXa7 and 24 bp for PthXo1) and the double-stranded DNA cleaving activity of FokI and, thus, are called TAL nucleases (TALNs). With all three TALNs, DNA is cleaved adjacent to the TAL-binding site under optimal conditions in vitro. When expressed in yeast, the TALNs promote DNA homologous recombination of a LacZ gene containing paired AvrXa7 or asymmetric AvrXa7/PthXo1 target sequences. Our results demonstrate the feasibility of creating a tool box of novel TALNs with potential for targeted genome modification in organisms lacking facile mechanisms for targeted gene knockout and homologous recombination.

  16. Prostaglandin D2-loaded microspheres effectively activate macrophage effector functions.

    PubMed

    Pereira, Priscilla Aparecida Tartari; Bitencourt, Claudia da Silva; dos Santos, Daiane Fernanda; Nicolete, Roberto; Gelfuso, Guilherme Martins; Faccioli, Lúcia Helena

    2015-10-12

    Biodegradable lactic-co-glycolic acid (PLGA) microspheres (MS) improve the stability of biomolecules stability and allow enable their sustained release. Lipid mediators represent a strategy for improving host defense; however, most of these mediators, such as prostaglandin D2 (PGD2), have low water solubility and are unstable. The present study aimed to develop and characterize MS loaded with PGD2 (PGD2-MS) to obtain an innovative tool to activate macrophages. PGD2-MS were prepared using an oil-in-water emulsion solvent extraction-evaporation process, and the size, zeta potential, surface morphology and encapsulation efficiency were determined. It was also evaluated in vitro the phagocytic index, NF-κB activation, as well as nitric oxide and cytokine production by alveolar macrophages (AMs) in response to PGD2-MS. PGD2-MS were spherical with a diameter of 5.0±3.3 μm and regular surface, zeta potential of -13.4±5.6 mV, and 36% of encapsulation efficiency, with 16-26% release of entrapped PGD2 at 4 and 48 h, respectively. PGD2-MS were more efficiently internalized by AMs than unloaded-MS, and activated NF-κB more than free PGD2. Moreover, PGD2-MS stimulated the production of nitric oxide, TNF-α, IL-1β, and TGF-β, more than free PGD2, indicating that microencapsulation increased the activating effect of PGD2 on cells. In LPS-pre-treated AMs, PGD2-MS decreased the release of IL-6 but increased the production of nitric oxide and IL-1β. These results show that the morphological characteristics of PGD2-MS facilitated interaction with, and activation of phagocytic cells; moreover, PGD2-MS retained the biological activities of PGD2 to trigger effector mechanisms in AMs. It is suggested that PGD2-MS represent a strategy for therapeutic intervention in the lungs of immunocompromised subjects.

  17. Cyclic AMP Effectors Regulate Myometrial Oxytocin Receptor Expression.

    PubMed

    Yulia, Angela; Singh, Natasha; Lei, Kaiyu; Sooranna, Suren R; Johnson, Mark R

    2016-11-01

    The factors that initiate human labor are poorly understood. We have tested the hypothesis that a decline in cAMP/protein kinase A (PKA) function leads to the onset of labor. Initially, we identified myometrial cAMP/PKA-responsive genes (six up-regulated and five down-regulated genes) and assessed their expression in myometrial samples taken from different stages of pregnancy and labor. We found that the oxytocin receptor (OTR) was one of the cAMP-repressed genes, and, given the importance of OTR in the labor process, we studied the mechanisms involved in greater detail using small interfering RNA, chemical agonists, and antagonists of the cAMP effectors. We found that cAMP-repressed genes, including OTR, increased with the onset of labor. Our in vitro studies showed that cAMP acting via PKA reduced OTR expression but that in the absence of PKA, cAMP acts via exchange protein activated by cAMP (EPAC) to increase OTR expression. In early labor myometrial samples, PKA levels and activity declined and Epac1 levels increased, perhaps accounting for the increase in myometrial OTR mRNA and protein levels at this time. In vitro exposure of myometrial cells to stretch and IL-1β increased OTR levels and reduced basal and forskolin-stimulated cAMP and PKA activity, as judged by phospho-cAMP response element-binding protein levels, but neither stretch nor IL-1β had any effect on PKA or EPAC1 levels. In summary, there is a reduction in the activity of the cAMP/PKA pathway with the onset of human labor potentially playing a critical role in regulating OTR expression and the transition from myometrial quiescence to activation.

  18. Changes in nucleoporin domain topology in response to chemical effectors.

    PubMed

    Paulillo, Sara M; Powers, Maureen A; Ullman, Katharine S; Fahrenkrog, Birthe

    2006-10-13

    Nucleoporins represent the molecular building blocks of nuclear pore complexes (NPCs), which mediate facilitated macromolecular trafficking between the cytoplasm and nucleus of eukaryotic cells. Phenylalanine-glycine (FG) repeat motifs are found in about one-third of the nucleoporins, and they provide major binding or docking sites for soluble transport receptors. We have shown recently that localization of the FG-repeat domains of vertebrate nucleoporins Nup153 and Nup214 within the NPC is influenced by its transport state. To test whether chemical effectors, such as calcium and ATP, influence the localization of the FG-repeat domains of Nup153 and Nup214 within the NPC, we performed immuno-electron microscopy of Xenopus oocyte nuclei using domain-specific antibodies against Nup153 and Nup214, respectively. Ca2+ and ATP are known to induce conformational changes in the NPC architecture, especially at the cytoplasmic face, but also at the nuclear basket of the NPC. We have found concentrations of calcium in the micromolar range or 1 mM ATP in the surrounding buffer leaves the spatial distribution of the FG-repeat of Nup153 and Nup214 largely unchanged. In contrast, ATP depletion, calcium store depletion by EGTA or thapsigargin, and high concentrations of divalent cation (i.e. 2 mM Ca2+ and 2 mM Mg2+) constrain the distribution of the FG-repeats of Nup153 and Nup214. Our data suggest that the location of the FG-repeat domains of Nup153 and Nup214 is sensitive to chemical changes within the near-field environment of the NPC.

  19. Structural consequences of effector protein complex formation in a diiron hydroxylase

    SciTech Connect

    Bailey, Lucas J.; McCoy, Jason G.; Phillips, Jr., George N.; Fox, Brian G.

    2009-06-12

    Carboxylate-bridged diiron hydroxylases are multicomponent enzyme complexes responsible for the catabolism of a wide range of hydrocarbons and as such have drawn attention for their mechanism of action and potential uses in bioremediation and enzymatic synthesis. These enzyme complexes use a small molecular weight effector protein to modulate the function of the hydroxylase. However, the origin of these functional changes is poorly understood. Here, we report the structures of the biologically relevant effector protein-hydroxylase complex of toluene 4-monooxygenase in 2 redox states. The structures reveal a number of coordinated changes that occur up to 25 {angstrom} from the active site and poise the diiron center for catalysis. The results provide a structural basis for the changes observed in a number of the measurable properties associated with effector protein binding. This description provides insight into the functional role of effector protein binding in all carboxylate-bridged diiron hydroxylases.

  20. End-effector: Joint conjugates for robotic assembly of large truss structures in space: Extended concepts

    NASA Technical Reports Server (NTRS)

    Brewer, W. V.; Rasis, E. P.; Shih, H. R.

    1993-01-01

    Results from NASA/HBCU Grant No. NAG-1-1125 are summarized. Designs developed for model fabrication, exploratory concepts drafted, interface of computer with robot and end-effector, and capability enhancement are discussed.

  1. Methods and Systems for Authorizing an Effector Command in an Integrated Modular Environment

    NASA Technical Reports Server (NTRS)

    Sunderland, Dean E. (Inventor); Ahrendt, Terry J. (Inventor); Moore, Tim (Inventor)

    2013-01-01

    Methods and systems are provided for authorizing a command of an integrated modular environment in which a plurality of partitions control actions of a plurality of effectors is provided. A first identifier, a second identifier, and a third identifier are determined. The first identifier identifies a first partition of the plurality of partitions from which the command originated. The second identifier identifies a first effector of the plurality of effectors for which the command is intended. The third identifier identifies a second partition of the plurality of partitions that is responsible for controlling the first effector. The first identifier and the third identifier are compared to determine whether the first partition is the same as the second partition for authorization of the command.

  2. The type three secreted effector SipC regulates the trafficking of PERP during Salmonella infection.

    PubMed

    Hallstrom, Kelly N; McCormick, Beth A

    2016-01-01

    Salmonella enterica Typhimurium employs type III secreted effectors to induce cellular invasion and pathogenesis. We previously reported the secreted effector SipA is in part responsible for inducing the apical accumulation of the host membrane protein PERP, a host factor we have shown is key to the inflammatory response induced by Salmonella. We now report that the S. Typhimurium type III secreted effector SipC significantly contributes to PERP redistribution to the apical membrane surface. To our knowledge, this is the first report demonstrating a role for SipC in directing the trafficking of a host membrane protein to the cell surface. In sum, facilitation of PERP trafficking appears to be a result of type III secreted effector-mediated recruitment of vesicles to the apical surface. Our study therefore reveals a new role for SipC, and builds upon previous reports suggesting recruitment of vesicles to the cell surface is important for Salmonella invasion.

  3. Functional domains and motifs of bacterial type III effector proteins and their roles in infection.

    PubMed

    Dean, Paul

    2011-11-01

    A key feature of the virulence of many bacterial pathogens is the ability to deliver effector proteins into eukaryotic cells via a dedicated type three secretion system (T3SS). Many bacterial pathogens, including species of Chlamydia, Xanthomonas, Pseudomonas, Ralstonia, Shigella, Salmonella, Escherichia and Yersinia, depend on the T3SS to cause disease. T3SS effectors constitute a large and diverse group of virulence proteins that mimic eukaryotic proteins in structure and function. A salient feature of bacterial effectors is their modular architecture, comprising domains or motifs that confer an array of subversive functions within the eukaryotic cell. These domains/motifs therefore represent a fascinating repertoire of molecular determinants with important roles during infection. This review provides a snapshot of our current understanding of bacterial effector domains and motifs where a defined role in infection has been demonstrated.

  4. Chloroplasts play a central role in plant defence and are targeted by pathogen effectors.

    PubMed

    de Torres Zabala, Marta; Littlejohn, George; Jayaraman, Siddharth; Studholme, David; Bailey, Trevor; Lawson, Tracy; Tillich, Michael; Licht, Dirk; Bölter, Bettina; Delfino, Laura; Truman, William; Mansfield, John; Smirnoff, Nicholas; Grant, Murray

    2015-06-01

    Microbe associated molecular pattern (MAMP) receptors in plants recognize MAMPs and activate basal defences; however a complete understanding of the molecular and physiological mechanisms conferring immunity remains elusive. Pathogens suppress active defence in plants through the combined action of effector proteins. Here we show that the chloroplast is a key component of early immune responses. MAMP perception triggers the rapid, large-scale suppression of nuclear encoded chloroplast-targeted genes (NECGs). Virulent Pseudomonas syringae effectors reprogramme NECG expression in Arabidopsis, target the chloroplast and inhibit photosynthetic CO2 assimilation through disruption of photosystem II. This activity prevents a chloroplastic reactive oxygen burst. These physiological changes precede bacterial multiplication and coincide with pathogen-induced abscisic acid (ABA) accumulation. MAMP pretreatment protects chloroplasts from effector manipulation, whereas application of ABA or the inhibitor of photosynthetic electron transport, DCMU, abolishes the MAMP-induced chloroplastic reactive oxygen burst, and enhances growth of a P. syringae hrpA mutant that fails to secrete effectors.

  5. Hitting the sweet spot-glycans as targets of fungal defense effector proteins.

    PubMed

    Künzler, Markus

    2015-05-06

    Organisms which rely solely on innate defense systems must combat a large number of antagonists with a comparably low number of defense effector molecules. As one solution of this problem, these organisms have evolved effector molecules targeting epitopes that are conserved between different antagonists of a specific taxon or, if possible, even of different taxa. In order to restrict the activity of the defense effector molecules to physiologically relevant taxa, these target epitopes should, on the other hand, be taxon-specific and easily accessible. Glycans fulfill all these requirements and are therefore a preferred target of defense effector molecules, in particular defense proteins. Here, we review this defense strategy using the example of the defense system of multicellular (filamentous) fungi against microbial competitors and animal predators.

  6. From GFP to β-lactamase: advancing intact cell imaging for toxins and effectors

    PubMed Central

    Zuverink, Madison; Barbieri, Joseph T.

    2015-01-01

    Canonical reporters such as green fluorescent protein (GFP) and luciferase have assisted researchers in probing cellular pathways and processes. Prior research in pathogenesis depended on sensitivity of biochemical and biophysical techniques to identify effectors and elucidate entry mechanisms. Recently, the β-lactamase (βlac) reporter system has advanced toxin and effector reporting by permitting measurement of βlac delivery into the cytosol or host βlac expression in intact cells. βlac measurement in cells was facilitated by the development of the fluorogenic substrate, CCF2-AM, to identify novel effectors, target cells, and domains involved in bacterial pathogenesis. The assay is also adaptable for high-throughput screening of small molecule inhibitors against toxins, providing information on mechanism and potential therapeutic agents. The versatility and limitations of the βlac reporter system as applied to toxins and effectors are discussed in this review. PMID:26500183

  7. Effectors of biotrophic fungi and oomycetes: pathogenicity factors and triggers of host resistance.

    PubMed

    Dodds, Peter N; Rafiqi, Maryam; Gan, Pamela H P; Hardham, Adrienne R; Jones, David A; Ellis, Jeffrey G

    2009-01-01

    Many biotrophic fungal and oomycete pathogens share a common infection process involving the formation of haustoria, which penetrate host cell walls and form a close association with plant membranes. Recent studies have identified a class of pathogenicity effector proteins from these pathogens that is transferred into host cells from haustoria during infection. This insight stemmed from the identification of avirulence (Avr) proteins from these pathogens that are recognized by intracellular host resistance (R) proteins. Oomycete effectors contain a conserved translocation motif that directs their uptake into host cells independently of the pathogen, and is shared with the human malaria pathogen. Genome sequence information indicates that oomycetes may express several hundred such host-translocated effectors. Elucidating the transport mechanism of fungal and oomycete effectors and their roles in disease offers new opportunities to understand how these pathogens are able to manipulate host cells to establish a parasitic relationship and to develop new disease-control measures.

  8. Human Effector / Initiator Gene Sets That Regulate Myometrial Contractility During Term and Preterm Labor

    PubMed Central

    WEINER, Carl P.; MASON, Clifford W.; DONG, Yafeng; BUHIMSCHI, Irina A.; SWAAN, Peter W.; BUHIMSCHI, Catalin S.

    2010-01-01

    Objective Distinct processes govern transition from quiescence to activation during term (TL) and preterm labor (PTL). We sought gene sets responsible for TL and PTL, along with the effector genes necessary for labor independent of gestation and underlying trigger. Methods Expression was analyzed in term and preterm +/− labor (n =6 subjects/group). Gene sets were generated using logic operations. Results 34 genes were similarly expressed in PTL/TL but absent from nonlabor samples (Effector Set). 49 genes were specific to PTL (Preterm Initiator Set) and 174 to TL (Term Initiator Set). The gene ontogeny processes comprising Term Initiator and Effector Sets were diverse, though inflammation was represented in 4 of the top 10; inflammation dominated the Preterm Initiator Set. Comments TL and PTL differ dramatically in initiator profiles. Though inflammation is part of the Term Initiator and the Effector Sets, it is an overwhelming part of PTL associated with intraamniotic inflammation. PMID:20452493

  9. Enhancement of Immune Effector Functions by Modulating IgG’s Intrinsic Affinity for Target Antigen

    PubMed Central

    Mazor, Yariv; Yang, Chunning; Borrok, M. Jack; Ayriss, Joanne; Aherne, Karen; Wu, Herren; Dall’Acqua, William F.

    2016-01-01

    Antibody-mediated immune effector functions play an essential role in the anti-tumor efficacy of many therapeutic mAbs. While much of the effort to improve effector potency has focused on augmenting the interaction between the antibody-Fc and activating Fc-receptors expressed on immune cells, the role of antibody binding interactions with the target antigen remains poorly understood. We show that antibody intrinsic affinity to the target antigen clearly influences the extent and efficiency of Fc-mediated effector mechanisms, and report the pivotal role of antibody binding valence on the ability to regulate effector functions. More particularly, we used an array of affinity modulated variants of three different mAbs, anti-CD4, anti-EGFR and anti-HER2 against a panel of target cell lines expressing disparate levels of the target antigen. We found that at saturating antibody concentrations, IgG variants with moderate intrinsic affinities, similar to those generated by the natural humoral immune response, promoted superior effector functions compared to higher affinity antibodies. We hypothesize that at saturating concentrations, effector function correlates most directly with the amount of Fc bound to the cell surface. Thus, high affinity antibodies exhibiting slow off-rates are more likely to interact bivalently with the target cell, occupying two antigen sites with a single Fc. In contrast, antibodies with faster off-rates are likely to dissociate each binding arm more rapidly, resulting in a higher likelihood of monovalent binding. Monovalent binding may in turn increase target cell opsonization and lead to improved recruitment of effector cells. This unpredicted relationship between target affinity and effector function potency suggests a careful examination of antibody design and engineering for the development of next-generation immunotherapeutics. PMID:27322177

  10. In-flight adaptive performance optimization (APO) control using redundant control effectors of an aircraft

    NASA Technical Reports Server (NTRS)

    Gilyard, Glenn B. (Inventor)

    1999-01-01

    Practical application of real-time (or near real-time) Adaptive Performance Optimization (APO) is provided for a transport aircraft in steady climb, cruise, turn descent or other flight conditions based on measurements and calculations of incremental drag from a forced response maneuver of one or more redundant control effectors defined as those in excess of the minimum set of control effectors required to maintain the steady flight condition in progress. The method comprises the steps of applying excitation in a raised-cosine form over an interval of from 100 to 500 sec. at the rate of 1 to 10 sets/sec of excitation, and data for analysis is gathered in sets of measurements made during the excitation to calculate lift and drag coefficients C.sub.L and C.sub.D from two equations, one for each coefficient. A third equation is an expansion of C.sub.D as a function of parasitic drag, induced drag, Mach and altitude drag effects, and control effector drag, and assumes a quadratic variation of drag with positions .delta..sub.i of redundant control effectors i=1 to n. The third equation is then solved for .delta..sub.iopt the optimal position of redundant control effector i, which is then used to set the control effector i for optimum performance during the remainder of said steady flight or until monitored flight conditions change by some predetermined amount as determined automatically or a predetermined minimum flight time has elapsed.

  11. Life, death and rebirth of avirulence effectors in a fungal pathogen of Brassica crops, Leptosphaeria maculans.

    PubMed

    Rouxel, Thierry; Balesdent, Marie-Hélène

    2017-04-01

    Contents 526 I. 526 II. 527 III. 527 IV. 529 V. 529 VI. 530 VII. 530 531 References 531 SUMMARY: In agricultural systems, major (R) genes for resistance in plants exert strong selection pressure on cognate/corresponding avirulence effector genes of phytopathogens. However, a complex interplay often exists between trade-offs linked to effector function and the need to escape R gene recognition. Here, using the Leptosphaeria maculans-oilseed rape pathosystem we review evolution of effectors submitted to multiple resistance gene selection. Characteristics of this pathosystem include a crop in which resistance genes have been deployed intensively resulting in 'boom and bust' cycles; a fungal pathogen with a high adaptive potential in which seven avirulence genes are cloned and for which population surveys have been coupled with molecular analysis of events responsible for virulence. The mode of evolution of avirulence genes, all located in dispensable parts of the 'two-speed' genome, is a highly dynamic gene-specific process. In some instances, avirulence genes are readily deleted under selection. However, others, even when located in the most plastic genome regions, undergo only limited point mutations or their avirulence phenotype is 'camouflaged' by another avirulence gene. Thus, while hundreds of effector genes are present, some effectors are likely to have an important and nonredundant function, suggesting functional redundancy and dispensability of effectors might not be the rule.

  12. Functional dissection of SseF, a membrane-integral effector protein of intracellular Salmonella enterica.

    PubMed

    Müller, Petra; Chikkaballi, Deepak; Hensel, Michael

    2012-01-01

    During intracellular life, the bacterial pathogen Salmonella enterica translocates a complex cocktail of effector proteins by means of the SPI2-encoded type III secretions system. The effectors jointly modify the endosomal system and vesicular transport in host cells. SseF and SseG are two effectors encoded by genes within Salmonella Pathogenicity Island 2 and both effector associate with endosomal membranes and microtubules and are involved in the formation of Salmonella-induced filaments. Our previous deletional analyses identified protein domains of SseF required for the effector function. Here we present a detailed mutational analysis that identifies a short hydrophobic motif as functionally essential. We demonstrate that SseF and SseG are still functional if translocated as a single fusion protein, but also mediate effector function if translocated in cells co-infected with sseF and sseG strains. SseF has characteristics of an integral membrane protein after translocation into host cells.

  13. bMERB domains are bivalent Rab8 family effectors evolved by gene duplication

    PubMed Central

    Rai, Amrita; Oprisko, Anastasia; Campos, Jeremy; Fu, Yangxue; Friese, Timon; Itzen, Aymelt; Goody, Roger S; Gazdag, Emerich Mihai; Müller, Matthias P

    2016-01-01

    In their active GTP-bound form, Rab proteins interact with proteins termed effector molecules. In this study, we have thoroughly characterized a Rab effector domain that is present in proteins of the Mical and EHBP families, both known to act in endosomal trafficking. Within our study, we show that these effectors display a preference for Rab8 family proteins (Rab8, 10, 13 and 15) and that some of the effector domains can bind two Rab proteins via separate binding sites. Structural analysis allowed us to explain the specificity towards Rab8 family members and the presence of two similar Rab binding sites that must have evolved via gene duplication. This study is the first to thoroughly characterize a Rab effector protein that contains two separate Rab binding sites within a single domain, allowing Micals and EHBPs to bind two Rabs simultaneously, thus suggesting previously unknown functions of these effector molecules in endosomal trafficking. DOI: http://dx.doi.org/10.7554/eLife.18675.001 PMID:27552051

  14. Functional versus effector-specific organization of the human posterior parietal cortex: revisited

    PubMed Central

    Leone, Frank T. M.; Medendorp, W. Pieter

    2016-01-01

    It has been proposed that the posterior parietal cortex (PPC) is characterized by an effector-specific organization. However, strikingly similar functional MRI (fMRI) activation patterns have been found in the PPC for hand and foot movements. Because the fMRI signal is related to average neuronal activity, similar activation levels may result either from effector-unspecific neurons or from intermingled subsets of effector-specific neurons within a voxel. We distinguished between these possibilities using fMRI repetition suppression (RS). Participants made delayed, goal-directed eye, hand, and foot movements to visual targets. In each trial, the instructed effector was identical or different to that of the previous trial. RS effects indicated an attenuation of the fMRI signal in repeat trials. The caudal PPC was active during the delay but did not show RS, suggesting that its planning activity was effector independent. Hand and foot-specific RS effects were evident in the anterior superior parietal lobule (SPL), extending to the premotor cortex, with limb overlap in the anterior SPL. Connectivity analysis suggested information flow between the caudal PPC to limb-specific anterior SPL regions and between the limb-unspecific anterior SPL toward limb-specific motor regions. These results underline that both function and effector specificity should be integrated into a concept of PPC action representation not only on a regional but also on a fine-grained, subvoxel level. PMID:27466132

  15. Effector-Mining in the Poplar Rust Fungus Melampsora larici-populina Secretome

    PubMed Central

    Lorrain, Cécile; Hecker, Arnaud; Duplessis, Sébastien

    2015-01-01

    The poplar leaf rust fungus, Melampsora larici-populina has been established as a tree-microbe interaction model. Understanding the molecular mechanisms controlling infection by pathogens appears essential for durable management of tree plantations. In biotrophic plant-parasites, effectors are known to condition host cell colonization. Thus, investigation of candidate secreted effector proteins (CSEPs) is a major goal in the poplar–poplar rust interaction. Unlike oomycetes, fungal effectors do not share conserved motifs and candidate prediction relies on a set of a priori criteria established from reported bona fide effectors. Secretome prediction, genome-wide analysis of gene families and transcriptomics of M. larici-populina have led to catalogs of more than a thousand secreted proteins. Automatized effector-mining pipelines hold great promise for rapid and systematic identification and prioritization of CSEPs for functional characterization. In this review, we report on and discuss the current status of the poplar rust fungus secretome and prediction of candidate effectors from this species. PMID:26697026

  16. SPRYSEC Effectors: A Versatile Protein-Binding Platform to Disrupt Plant Innate Immunity

    PubMed Central

    Diaz-Granados, Amalia; Petrescu, Andrei-José; Goverse, Aska; Smant, Geert

    2016-01-01

    Persistent infections by sedentary plant-parasitic nematodes are a major threat to important food crops all over the world. These roundworms manipulate host plant cell morphology and physiology to establish sophisticated feeding structures. Key modifications to plant cells during their transition into feeding structures are largely attributed to the activity of effectors secreted by the nematodes. The SPRYSEC effectors were initially identified in the potato cyst nematodes Globodera rostochiensis and G. pallida, and are characterized by a single SPRY domain, a non-catalytic domain present in modular proteins with different functions. The SPRY domain is wide-spread among eukaryotes and thought to be involved in mediating protein–protein interactions. Thus far, the SPRY domain is only reported as a functional domain in effectors of plant-parasitic nematodes, but not of other plant pathogens. SPRYSEC effectors have been implicated in both suppression and activation of plant immunity, but other possible roles in nematode virulence remain undefined. Here, we review the latest reports on the structure, function, and sequence diversity of SPRYSEC effectors, which provide support for a model featuring these effectors as a versatile protein-binding platform for the nematodes to target a wide range of host proteins during parasitism. PMID:27812363

  17. Computational prediction of type III and IV secreted effectors in Gram-negative bacteria

    SciTech Connect

    McDermott, Jason E.; Corrigan, Abigail L.; Peterson, Elena S.; Oehmen, Christopher S.; Niemann, George; Cambronne, Eric; Sharp, Danna; Adkins, Joshua N.; Samudrala, Ram; Heffron, Fred

    2011-01-01

    In this review, we provide an overview of the methods employed by four recent papers that described novel methods for computational prediction of secreted effectors from type III and IV secretion systems in Gram-negative bacteria. The results of the studies in terms of performance at accurately predicting secreted effectors and similarities found between secretion signals that may reflect biologically relevant features for recognition. We discuss the web-based tools for secreted effector prediction described in these studies and announce the availability of our tool, the SIEVEserver (http://www.biopilot.org). Finally, we assess the accuracy of the three type III effector prediction methods on a small set of proteins not known prior to the development of these tools that we have recently discovered and validated using both experimental and computational approaches. Our comparison shows that all methods use similar approaches and, in general arrive at similar conclusions. We discuss the possibility of an order-dependent motif in the secretion signal, which was a point of disagreement in the studies. Our results show that there may be classes of effectors in which the signal has a loosely defined motif, and others in which secretion is dependent only on compositional biases. Computational prediction of secreted effectors from protein sequences represents an important step toward better understanding the interaction between pathogens and hosts.

  18. Additive roles of PthAs in bacterial growth and pathogenicity associated with nucleotide polymorphisms in effector-binding elements of citrus canker susceptibility genes.

    PubMed

    Abe, Valeria Yukari; Benedetti, Celso Eduardo

    2016-10-01

    Citrus canker, caused by Xanthomonas citri, affects most commercial citrus varieties. All X. citri strains possess at least one transcription activator-like effector of the PthA family that activates host disease susceptibility (S) genes. The X. citri strain 306 encodes four PthA effectors; nevertheless, only PthA4 is known to elicit cankers on citrus. As none of the PthAs act as avirulence factors on citrus, we hypothesized that PthAs 1-3 might also contribute to pathogenicity on certain hosts. Here, we show that, although PthA4 is indispensable for canker formation in six Brazilian citrus varieties, PthAs 1 and 3 contribute to canker development in 'Pera' sweet orange, but not in 'Tahiti' lemon. Deletions in two or more pthA genes reduce bacterial growth in planta more pronouncedly than single deletions, suggesting an additive role of PthAs in pathogenicity and bacterial fitness. The contribution of PthAs 1 and 3 in canker formation in 'Pera' plants does not correlate with the activation of the canker S gene, LOB1 (LATERAL ORGAN BOUNDARIES 1), but with the induction of other PthA targets, including LOB2 and citrus dioxygenase (DIOX). LOB1, LOB2 and DIOX show differential PthA-dependent expression between 'Pera' and 'Tahiti' plants that appears to be associated with nucleotide polymorphisms found at or near PthA-binding sites. We also present evidence that LOB1 activation alone is not sufficient to elicit cankers on citrus, and that DIOX acts as a canker S gene in 'Pera', but not 'Tahiti', plants. Our results suggest that the activation of multiple S genes, such as LOB1 and DIOX, is necessary for full canker development.

  19. Escape from Management Hell: 12 Tales of Horror, Humor, and Heroism.

    ERIC Educational Resources Information Center

    Gilbreath, Robert D.

    This book offers a set of stories in which corporate executives demonstrate the folly and futility of their own business practices. In the stories, 12 executives are trying to escape from a hell of their own making. The tales provide insights into the management woes with which people at all levels deal on a daily basis. Topics include: the…

  20. Feigning terminal illness to get narcotics: a cautionary tale for hospices.

    PubMed

    Gonzalez, Faustino; Galante, Mirta

    2012-08-01

    We present the case of a woman who enrolled in the hospice benefit in order to obtain narcotics. We believe this is a cautionary tale for hospices because of our propensity to enroll patients with minimal corroborating information, in order not to delay symptom management. Also we are philosophically predisposed to believe a patient's self-report of pain and other distressing symptoms.