Full Text Available An enhanced understanding of the hookworm genome and its resident mobile genetic elements should facilitate understanding of the genome evolution, genome organization, possibly host-parasite co-evolution and horizontal gene transfer, and from a practical perspective, development of transposon-based transgenesis for hookworms and other parasitic nematodes.A novel mariner-like element (MLE was characterized from the genome of the dog hookworm, Ancylostoma caninum, and termed bandit. The consensus sequence of the bandit transposon was 1,285 base pairs (bp in length. The new transposon was flanked by perfect terminal inverted repeats of 32 nucleotides in length with a common target site duplication TA, and it encoded an open reading frame (ORF of 342 deduced amino acid residues. Phylogenetic comparisons confirmed that the ORF encoded a mariner-like transposase, which included conserved catalytic domains, and that the bandit transposon belonged to the cecropia subfamily of MLEs. The phylogenetic analysis also indicated that the Hsmar1 transposon from humans was the closest known relative of bandit, and that bandit and Hsmar1 constituted a clade discrete from the Tc1 subfamily of MLEs from the nematode Caenorhabditis elegans. Moreover, homology models based on the crystal structure of Mos1 from Drosophila mauritiana revealed closer identity in active site residues of the catalytic domain including Ser281, Lys289 and Asp293 between bandit and Hsmar1 than between Mos1 and either bandit or Hsmar1. The entire bandit ORF was amplified from genomic DNA and a fragment of the bandit ORF was amplified from RNA, indicating that this transposon is actively transcribed in hookworms.A mariner-like transposon termed bandit has colonized the genome of the hookworm A. caninum. Although MLEs exhibit a broad host range, and are identified in other nematodes, the closest phylogenetic relative of bandit is the Hsmar1 element of humans. This surprising finding suggests
Rostovskaya, Maria; Fu, Jun; Obst, Mandy; Baer, Isabell; Weidlich, Stefanie; Wang, Hailong; Smith, Andrew J. H.; Anastassiadis, Konstantinos; Stewart, A. Francis
Transgenesis is a cornerstone of molecular biology. The ability to integrate a specifically engineered piece of DNA into the genome of a living system is fundamental to our efforts to understand life and exploit its implications for medicine, nanotechnology and bioprospecting. However, transgenesis has been hampered by position effects and multi-copy integration problems, which are mainly due to the use of small, plasmid-based transgenes. Large transgenes based on native genomic regions cloned into bacterial artificial chromosomes (BACs) circumvent these problems but are prone to fragmentation. Herein, we report that contrary to widely held notions, large BAC-sized constructs do not prohibit transposition. We also report the first reliable method for BAC transgenesis in human embryonic stem cells (hESCs). The PiggyBac or Sleeping Beauty transposon inverted repeats were integrated into BAC vectors by recombineering, followed by co-lipofection with the corresponding transposase in hESCs to generate robust fluorescent protein reporter lines for OCT4, NANOG, GATA4 and PAX6. BAC transposition delivers several advantages, including increased frequencies of single-copy, full-length integration, which will be useful in all transgenic systems but especially in difficult venues like hESCs. PMID:22753106
Staunstrup, Nicklas H; Moldt, Brian; Mátés, Lajos
Gene delivery by human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (LVs) is efficient, but genomic integration of the viral DNA is strongly biased toward transcriptionally active loci resulting in an increased risk of insertional mutagenesis in gene therapy protocols. Nonviral...... Sleeping Beauty (SB) transposon vectors have a significantly safer insertion profile, but efficient delivery into relevant cell/tissue types is a limitation. In an attempt to combine the favorable features of the two vector systems we established a novel hybrid vector technology based on SB transposase......-mediated insertion of lentiviral DNA circles generated during transduction of target cells with integrase (IN)-defective LVs (IDLVs). By construction of a lentivirus-transposon hybrid vector allowing transposition exclusively from circular viral DNA substrates, we demonstrate that SB transposase added in trans...
Xue, Chi; Goldenfeld, Nigel
Transposable elements, or transposons, are DNA sequences that can jump from site to site in the genome during the life cycle of a cell, usually encoding the very enzymes which perform their excision. However, some transposons are parasitic, relying on the enzymes produced by the regular transposons. In this case, we show that a stochastic model, which takes into account the small copy numbers of the active transposons in a cell, predicts noise-induced predator-prey oscillations with a characteristic time scale that is much longer than the cell replication time, indicating that the state of the predator-prey oscillator is stored in the genome and transmitted to successive generations. Our work demonstrates the important role of the number fluctuations in the expression of mobile genetic elements, and shows explicitly how ecological concepts can be applied to the dynamics and fluctuations of living genomes.
Warburton, Philip J; Allan, Elaine; Hunter, Stephanie; Ward, John; Booth, Veronica; Wade, William G; Mullany, Peter
The human oral cavity is host to a complex microbial community estimated to comprise >700 bacterial species, of which at least half are thought to be not yet cultivable in vitro. To investigate the plasmids present in this community, we used a transposon-aided capture system, which allowed the isolation of plasmids from human oral supra- and subgingival plaque samples. Thirty-two novel plasmids and a circular molecule that could be an integrase-generated circular intermediate were isolated. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
de Vries, Lisbeth Elvira; Christensen, H.; Skov, R. L.
To analyse the sequence diversity of the tetracycline resistance gene tet(M) in Staphylococcus aureus from humans and animals and to determine mobile elements associated with tet(M) in S. aureus. In total, 205 tetracycline-resistant isolates were screened for tet(M) by PCR. tet(M) genes were...... sequenced and compared with tet(M) deposited in GenBank. Based on phylogenetic analysis isolates were screened for Tn916- and Tn5801-like xis/int genes, and transposons were confirmed by linking PCR. spa typing was performed and selected isolates were used as donors in a filter mating experiment. Forty......-one isolates (21.3%, 60.7%, 2.6% and 4.4% of the human, pig, poultry and cattle isolates, respectively) were tet(M) positive. tet(M) was located on Tn5801-like and Tn916-like transposons in humans and on a specific Tn916-like element in animals. Human isolates were of different spa types (t034, t008, t037, t...
Siegrist, M Sloan; Rubin, Eric J
Phage transduction is an attractive method of genetic manipulation in mycobacteria. PhiMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an Escherichia coli oriR6 K origin of replication. Mycobacterial transposon mutant libraries produced by PhiMycoMarT7 transduction are amenable to both forward and reverse genetic studies. In this protocol, we detail the preparation of PhiMycoMarT7, including a description of the phage, reconstitution of the phage, purification of plaques, preparation of phage stock, and titering of phage stock. We then describe the transduction procedure and finally outline the isolation of individual transposon mutants.
Full Text Available Transposable genetic elements are ubiquitous, yet their presence or absence at any given position within a genome can vary between individual cells, tissues, or strains. Transposable elements have profound impacts on host genomes by altering gene expression, assisting in genomic rearrangements, causing insertional mutations, and serving as sources of phenotypic variation. Characterizing a genome's full complement of transposons requires whole genome sequencing, precluding simple studies of the impact of transposition on interindividual variation. Here, we describe a global mapping approach for identifying transposon locations in any genome, using a combination of transposon-specific DNA extraction and microarray-based comparative hybridization analysis. We use this approach to map the repertoire of endogenous transposons in different laboratory strains of Saccharomyces cerevisiae and demonstrate that transposons are a source of extensive genomic variation. We also apply this method to mapping bacterial transposon insertion sites in a yeast genomic library. This unique whole genome view of transposon location will facilitate our exploration of transposon dynamics, as well as defining bases for individual differences and adaptive potential.
Staunstrup, Nicklas Heine; Madsen, Johannes; Primo, Maria Nascimento; Li, Juan; Liu, Ying; Kragh, Peter M.; Li, Rong; Schmidt, Mette; Purup, Stig; Dagnæs-Hansen, Frederik; Svensson, Lars; Petersen, Thomas K.; Callesen, Henrik; Bolund, Lars; Mikkelsen, Jacob Giehm
Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage
Carlson Daniel F
Full Text Available Abstract Background Swine is an important agricultural commodity and biomedical model. Manipulation of the pig genome provides opportunity to improve production efficiency, enhance disease resistance, and add value to swine products. Genetic engineering can also expand the utility of pigs for modeling human disease, developing clinical treatment methodologies, or donating tissues for xenotransplantation. Realizing the full potential of pig genetic engineering requires translation of the complete repertoire of genetic tools currently employed in smaller model organisms to practical use in pigs. Results Application of transposon and recombinase technologies for manipulation of the swine genome requires characterization of their activity in pig cells. We tested four transposon systems- Sleeping Beauty, Tol2, piggyBac, and Passport in cultured porcine cells. Transposons increased the efficiency of DNA integration up to 28-fold above background and provided for precise delivery of 1 to 15 transgenes per cell. Both Cre and Flp recombinase were functional in pig cells as measured by their ability to remove a positive-negative selection cassette from 16 independent clones and over 20 independent genomic locations. We also demonstrated a Cre-dependent genetic switch capable of eliminating an intervening positive-negative selection cassette and activating GFP expression from episomal and genome-resident transposons. Conclusion We have demonstrated for the first time that transposons and recombinases are capable of mobilizing DNA into and out of the porcine genome in a precise and efficient manner. This study provides the basis for developing transposon and recombinase based tools for genetic engineering of the swine genome.
Hackett, Perry B.; Largaespada, David A.; Switzer, Kirsten C.; Cooper, Laurence J.N.
Investigational therapy can be successfully undertaken using viral- and non-viral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR+ T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease. PMID:23313630
Full Text Available Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. 'Cut and paste' DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB and piggyBac (PB that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1, a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB.
Ruiz, Lorena; Motherway, Mary O'Connell; Lanigan, Noreen; van Sinderen, Douwe
Bifidobacteria are claimed to contribute positively to human health through a range of beneficial or probiotic activities, including amelioration of gastrointestinal and metabolic disorders, and therefore this particular group of gastrointestinal commensals has enjoyed increasing industrial and scientific attention in recent years. However, the molecular mechanisms underlying these probiotic mechanisms are still largely unknown, mainly due to the fact that molecular tools for bifidobacteria are rather poorly developed, with many strains lacking genetic accessibility. In this work, we describe the generation of transposon insertion mutants in two bifidobacterial strains, B. breve UCC2003 and B. breve NCFB2258. We also report the creation of the first transposon mutant library in a bifidobacterial strain, employing B. breve UCC2003 and a Tn5-based transposome strategy. The library was found to be composed of clones containing single transposon insertions which appear to be randomly distributed along the genome. The usefulness of the library to perform phenotypic screenings was confirmed through identification and analysis of mutants defective in D-galactose, D-lactose or pullulan utilization abilities.
Full Text Available Bifidobacteria are claimed to contribute positively to human health through a range of beneficial or probiotic activities, including amelioration of gastrointestinal and metabolic disorders, and therefore this particular group of gastrointestinal commensals has enjoyed increasing industrial and scientific attention in recent years. However, the molecular mechanisms underlying these probiotic mechanisms are still largely unknown, mainly due to the fact that molecular tools for bifidobacteria are rather poorly developed, with many strains lacking genetic accessibility. In this work, we describe the generation of transposon insertion mutants in two bifidobacterial strains, B. breve UCC2003 and B. breve NCFB2258. We also report the creation of the first transposon mutant library in a bifidobacterial strain, employing B. breve UCC2003 and a Tn5-based transposome strategy. The library was found to be composed of clones containing single transposon insertions which appear to be randomly distributed along the genome. The usefulness of the library to perform phenotypic screenings was confirmed through identification and analysis of mutants defective in D-galactose, D-lactose or pullulan utilization abilities.
Ivics, Zoltán; Izsvák, Zsuzsanna
Transposable elements can be viewed as natural DNA transfer vehicles that, similar to integrating viruses, are capable of efficient genomic insertion. The mobility of class II transposable elements (DNA transposons) can be controlled by conditionally providing the transposase component of the transposition reaction. Thus, a DNA of interest (be it a fluorescent marker, a small hairpin (sh)RNA expression cassette, a mutagenic gene trap or a therapeutic gene construct) cloned between the inverted repeat sequences of a transposon-based vector can be used for stable genomic insertion in a regulated and highly efficient manner. This methodological paradigm opened up a number of avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture, the production of germline transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species, and therapy of genetic disorders in humans. Sleeping Beauty (SB) was the first transposon shown to be capable of gene transfer in vertebrate cells, and recent results confirm that SB supports a full spectrum of genetic engineering including transgenesis, insertional mutagenesis, and therapeutic somatic gene transfer both ex vivo and in vivo. The first clinical application of the SB system will help to validate both the safety and efficacy of this approach. In this review, we describe the major transposon systems currently available (with special emphasis on SB), discuss the various parameters and considerations pertinent to their experimental use, and highlight the state of the art in transposon technology in diverse genetic applications.
Tellier, Michael; Bouuaert, Corentin Claeys; Chalmers, Ronald
The IS630-Tc1-mariner (ITm) family of transposons is one of the most widespread in nature. The phylogenetic distribution of its members shows that they do not persist for long in a given lineage, but rely on frequent horizontal transfer to new hosts. Although they are primarily selfish genomic-parasites, ITm transposons contribute to the evolution of their hosts because they generate variation and contribute protein domains and regulatory regions. Here we review the molecular mechanism of ITm transposition and its regulation. We focus mostly on the mariner elements, which are understood in the greatest detail owing to in vitro reconstitution and structural analysis. Nevertheless, the most important characteristics are probably shared across the grouping. Members of the ITm family are mobilized by a cut-and-paste mechanism and integrate at 5'-TA dinucleotide target sites. The elements encode a single transposase protein with an N-terminal DNA-binding domain and a C-terminal catalytic domain. The phosphoryl-transferase reactions during the DNA-strand breaking and joining reactions are performed by the two metal-ion mechanism. The metal ions are coordinated by three or four acidic amino acid residues located within an RNase H-like structural fold. Although all of the strand breaking and joining events at a given transposon end are performed by a single molecule of transposase, the reaction is coordinated by close communication between transpososome components. During transpososome assembly, transposase dimers compete for free transposon ends. This helps to protect the host by dampening an otherwise exponential increase in the rate of transposition as the copy number increases.
Full Text Available Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700-6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.
Kebriaei, Partow; Izsvák, Zsuzsanna; Narayanavari, Suneel A; Singh, Harjeet; Ivics, Zoltán
The widespread clinical implementation of gene therapy requires the ability to stably integrate genetic information through gene transfer vectors in a safe, effective, and economical manner. The latest generation of Sleeping Beauty (SB) transposon vectors fulfills these requirements, and may overcome limitations associated with viral gene transfer vectors and transient nonviral gene delivery approaches that are prevalent in ongoing clinical trials. The SB system enables high-level stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, thereby representing a highly attractive gene transfer strategy for clinical use. Here, we review the most important aspects of using SB for gene therapy, including vectorization as well as genomic integration features. We also illustrate the path to successful clinical implementation by highlighting the application of chimeric antigen receptor (CAR)-modified T cells in cancer immunotherapy. Copyright © 2017 Elsevier Ltd. All rights reserved.
Full Text Available Abstract Transposable elements can be viewed as natural DNA transfer vehicles that, similar to integrating viruses, are capable of efficient genomic insertion. The mobility of class II transposable elements (DNA transposons can be controlled by conditionally providing the transposase component of the transposition reaction. Thus, a DNA of interest (be it a fluorescent marker, a small hairpin (shRNA expression cassette, a mutagenic gene trap or a therapeutic gene construct cloned between the inverted repeat sequences of a transposon-based vector can be used for stable genomic insertion in a regulated and highly efficient manner. This methodological paradigm opened up a number of avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture, the production of germline transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species, and therapy of genetic disorders in humans. Sleeping Beauty (SB was the first transposon shown to be capable of gene transfer in vertebrate cells, and recent results confirm that SB supports a full spectrum of genetic engineering including transgenesis, insertional mutagenesis, and therapeutic somatic gene transfer both ex vivo and in vivo. The first clinical application of the SB system will help to validate both the safety and efficacy of this approach. In this review, we describe the major transposon systems currently available (with special emphasis on SB, discuss the various parameters and considerations pertinent to their experimental use, and highlight the state of the art in transposon technology in diverse genetic applications.
Nilsson, Martin; Christiansen, Natalia; Høiby, Niels
This article describes the construction and characterization of a mariner-based transposon vector designed for use in oral streptococci, but with a potential use in other Gram-positive bacteria. The new transposon vector, termed pMN100, contains the temperature-sensitive origin of replication rep...... 5000 mutants was used in a screen to identify genes involved in the production of sucrose-dependent extracellular matrix components. Mutants with transposon inserts in genes encoding glycosyltransferases and the competence-related secretory locus were predominantly found in this screen....
. Abstract. Mobilization of two P element subfamilies (canonical and O-type) from Drosophila sturtevanti and D. saltans was evaluated for copy number and transposition activity using the transposon display (TD) technique. Pairwise distances ...
Gnerer, Joshua P; Venken, Koen J T; Dierick, Herman A
Binary expression systems such as GAL4/UAS, LexA/LexAop and QF/QUAS have greatly enhanced the power of Drosophila as a model organism by allowing spatio-temporal manipulation of gene function as well as cell and neural circuit function. Tissue-specific expression of these heterologous transcription factors relies on random transposon integration near enhancers or promoters that drive the binary transcription factor embedded in the transposon. Alternatively, gene-specific promoter elements are directly fused to the binary factor within the transposon followed by random or site-specific integration. However, such insertions do not consistently recapitulate endogenous expression. We used Minos-Mediated Integration Cassette (MiMIC) transposons to convert host loci into reliable gene-specific binary effectors. MiMIC transposons allow recombinase-mediated cassette exchange to modify the transposon content. We developed novel exchange cassettes to convert coding intronic MiMIC insertions into gene-specific binary factor protein-traps. In addition, we expanded the set of binary factor exchange cassettes available for non-coding intronic MiMIC insertions. We show that binary factor conversions of different insertions in the same locus have indistinguishable expression patterns, suggesting that they reliably reflect endogenous gene expression. We show the efficacy and broad applicability of these new tools by dissecting the cellular expression patterns of the Drosophila serotonin receptor gene family. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
Characterization of the Pathogenicity of Streptococcus intermedius TYG1620 Isolated from a Human Brain Abscess Based on the Complete Genome Sequence with Transcriptome Analysis and Transposon Mutagenesis in a Murine Subcutaneous Abscess Model.
Hasegawa, Noriko; Sekizuka, Tsuyoshi; Sugi, Yutaka; Kawakami, Nobuhiro; Ogasawara, Yumiko; Kato, Kengo; Yamashita, Akifumi; Takeuchi, Fumihiko; Kuroda, Makoto
Streptococcus intermedius is known to cause periodontitis and pyogenic infections in the brain and liver. Here we report the complete genome sequence of strain TYG1620 (genome size, 2,006,877 bp; GC content, 37.6%; 2,020 predicted open reading frames [ORFs]) isolated from a brain abscess in an infant. Comparative analysis of S. intermedius genome sequences suggested that TYG1620 carries a notable type VII secretion system (T7SS), two long repeat regions, and 19 ORFs for cell wall-anchored proteins (CWAPs). To elucidate the genes responsible for the pathogenicity of TYG1620, transcriptome analysis was performed in a murine subcutaneous abscess model. The results suggest that the levels of expression of small hypothetical proteins similar to phenol-soluble modulin β1 (PSMβ1), a staphylococcal virulence factor, significantly increased in the abscess model. In addition, an experiment in a murine subcutaneous abscess model with random transposon (Tn) mutant attenuation suggested that Tn mutants with mutations in 212 ORFs in the Tn mutant library were attenuated in the murine abscess model (629 ORFs were disrupted in total); the 212 ORFs are putatively essential for abscess formation. Transcriptome analysis identified 37 ORFs, including paralogs of the T7SS and a putative glucan-binding CWAP in long repeat regions, to be upregulated and attenuated in vivo This study provides a comprehensive characterization of S. intermedius pathogenicity based on the complete genome sequence and a murine subcutaneous abscess model with transcriptome and Tn mutagenesis, leading to the identification of pivotal targets for vaccines or antimicrobial agents for the control of S. intermedius infections. Copyright © 2017 American Society for Microbiology.
Tsukahara, T; Iwase, N; Kawakami, K; Iwasaki, M; Yamamoto, C; Ohmine, K; Uchibori, R; Teruya, T; Ido, H; Saga, Y; Urabe, M; Mizukami, H; Kume, A; Nakamura, M; Brentjens, R; Ozawa, K
Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.
Aboklaish, Ali F; Dordet-Frisoni, Emilie; Citti, Christine; Toleman, Mark A; Glass, John I; Spiller, O Brad
While transposon mutagenesis has been successfully used for Mycoplasma spp. to disrupt and determine non-essential genes, previous attempts with Ureaplasma spp. have been unsuccessful. Using a polyethylene glycol-transformation enhancing protocol, we were able to transform three separate serovars of Ureaplasma parvum with a Tn4001-based mini-transposon plasmid containing a gentamicin resistance selection marker. Despite the large degree of homology between Ureaplasma parvum and Ureaplasma urealyticum, all attempts to transform the latter in parallel failed, with the exception of a single clinical U. urealyticum isolate. PCR probing and sequencing were used to confirm transposon insertion into the bacterial genome and identify disrupted genes. Transformation of prototype serovar 3 consistently resulted in transfer only of sequence between the mini-transposon inverted repeats, but some strains showed additional sequence transfer. Transposon insertion occurred randomly in the genome resulting in unique disruption of genes UU047, UU390, UU440, UU450, UU520, UU526, UU582 for single clones from a panel of screened clones. An intergenic insertion between genes UU187 and UU188 was also characterised. Two phenotypic alterations were observed in the mutated strains: Disruption of a DEAD-box RNA helicase (UU582) altered growth kinetics, while the U. urealyticum strain lost resistance to serum attack coincident with disruption of gene UUR10_137 and loss of expression of a 41 kDa protein. Transposon mutagenesis was used successfully to insert single copies of a mini-transposon into the genome and disrupt genes leading to phenotypic changes in Ureaplasma parvum strains. This method can now be used to deliver exogenous genes for expression and determine essential genes for Ureaplasma parvum replication in culture and experimental models. Copyright © 2014 Elsevier GmbH. All rights reserved.
Haring, M; Veken, J; Windrich, R; Kneppers, T; Rommens, C; Nijkamp, H J.J.; Hille, J [Department of Genetics, Free University, Amsterdam (Netherlands)
Full text: The discovery of mobile genetic elements in plants has permitted the use of these transposons for insertional mutagenesis. This applies so far only to Zea mays and Antirrhinum majus, because other plant transposable elements have not been characterised so thoroughly at the genetic and the molecular level. To establish whether transposons (Ac from maize and Tam3 from Antirrhinum) remain mobile in heterologous hosts, either in somatic tissue or after meiosis, a phenotypic assay system for transposition was developed. The separation of the two transposition functions will allow controlled mutagenesis of plant genes. Our results indicate that both transposable elements remain active in heterologous hosts. (author)
Full Text Available Aggregatibacter actinomycetemcomitans is the primary etiological agent of aggressive periodontal disease. Identification of novel virulence factors at the genome-wide level is hindered by lack of efficient genetic tools to perform mutagenesis in this organism. The Himar1 mariner transposon is known to yield a random distribution of insertions in an organism’s genome with requirement for only a TA dinucleotide target and is independent of host-specific factors. However, the utility of this system in A. actinomycetemcomitans is unknown. In this study, we found that Himar1 transposon mutagenesis occurs at a high frequency (×10-4, and can be universally applied to wild-type A. actinomycetemcomitans strains of serotypes a, b, and c. The Himar1 transposon inserts were stably inherited in A. actinomycetemcomitans transconjugants in the absence of antibiotics. A library of 16,000 mutant colonies of A. actinomycetemcomitans was screened for reduced biofilm formation. Mutants with transposon inserts in genes encoding pilus, putative ion transporters, multidrug resistant proteins, transcription regulators and enzymes involved in the synthesis of extracellular polymeric substance, bacterial metabolism and stress response were discovered in this screen. Our results demonstrated the utility of the Himar1 mutagenesis system as a novel genetic tool for functional genomic analysis in A. actinomycetemcomitans.
Nouroz, F.; Noreen, S.; Harrison, J.S.H.
Transposable elements are the major drivers of genome evolution and plasticity. Due to their transposition mode, they are classified into two major classes as Retrotransposons and DNA transposons. The En/Spm or CACTA elements are diverse group of DNA transposons proliferating in plant genomes. Various bioinformatics and molecular approaches were used for identification and distribution of CACTA transposons in Brassica genome. A combination of dot plot analysis and BLASTN searches yielded 35 autonomous and 7 non-autonomous CACTA elements in Brassica. The elements ranged in sizes from 1.2 kb non-autonomous elements to 11kb autonomous elements, terminated by 3 bp Target Site Duplication (TSD) and ~15 bp conserved Terminal Inverted Repeat (TIR) motifs (5'-CACTACAAGAAAACA-3'), with heterogeneous internal regions. The transposase (TNP) was identified from autonomous CACTA elements, while other protein domains from Brassica and other plants CACTA revealed similar organizations with minor differences. Both transposases (TNPD, TNPA) are present in most CACTA, while a few CACTA harboured an additional ATHILA ORF1-like domain. The PCR analysis amplified the CACTA transposases from 40 Brassica accessions (A, B, and C-genome) suggesting their distribution among various Brassica crops. A detailed characterization and evolutionary analysis of the identified CACTA elements allowed some to be placed in genome-specific groups, while most of them (Brassica-Arabidopsis elements) have followed the same evolutionary line. The distribution of CACTA in Brassica concluded that 3 bp TSDs generating CACTA transposons contributed significantly to genome size and evolution of Brassica genome. (author)
Full Text Available Ciliated protists rearrange their genomes dramatically during nuclear development via chromosome fragmentation and DNA deletion to produce a trimmer and highly reorganized somatic genome. The deleted portion of the genome includes potentially active transposons or transposon-like sequences that reside in the germline. Three independent studies recently showed that transposase proteins of the DDE/DDD superfamily are indispensible for DNA processing in three distantly related ciliates. In the spirotrich Oxytricha trifallax, high copy-number germline-limited transposons mediate their own excision from the somatic genome but also contribute to programmed genome rearrangement through a remarkable transposon mutualism with the host. By contrast, the genomes of two oligohymenophorean ciliates, Tetrahymena thermophila and Paramecium tetraurelia, encode homologous PiggyBac-like transposases as single-copy genes in both their germline and somatic genomes. These domesticated transposases are essential for deletion of thousands of different internal sequences in these species. This review contrasts the events underlying somatic genome reduction in three different ciliates and considers their evolutionary origins and the relationships among their distinct mechanisms for genome remodeling.
Kawakami, Koichi; Largaespada, David A; Ivics, Zoltán
Genetic tools and mutagenesis strategies based on transposable elements are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of their inherent capacity to insert into DNA, transposons can be developed into powerful tools for chromosomal manipulations. Transposon-based forward mutagenesis screens have numerous advantages including high throughput, easy identification of mutated alleles, and providing insight into genetic networks and pathways based on phenotypes. For example, the Sleeping Beauty transposon has become highly instrumental to induce tumors in experimental animals in a tissue-specific manner with the aim of uncovering the genetic basis of diverse cancers. Here, we describe a battery of mutagenic cassettes that can be applied in conjunction with transposon vectors to mutagenize genes, and highlight versatile experimental strategies for the generation of engineered chromosomes for loss-of-function as well as gain-of-function mutagenesis for functional gene annotation in vertebrate models, including zebrafish, mice, and rats. Copyright © 2017 Elsevier Ltd. All rights reserved.
Hackett Perry B
Full Text Available Abstract Background Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern. Results Here we report the systematic development and testing of a transposon-based gene-trap system incorporating the doxycycline-repressible Tet-Off (tTA system that is capable of activating the expression of genes under control of a Tet response element (TRE promoter. We demonstrate that the gene trap system is fully functional in vitro by introducing the "gene-trap tTA" vector into human cells by transposition and identifying clones that activate expression of a TRE-luciferase transgene in a doxycycline-dependent manner. In transgenic mice, we mobilize gene-trap tTA vectors, discover parameters that can affect germline mobilization rates, and identify candidate gene insertions to demonstrate the in vivo functionality of the vector system. We further demonstrate that the gene-trap can act as a reporter of endogenous gene expression and it can be coupled with bioluminescent imaging to identify genes with tissue-specific expression patterns. Conclusion Akin to the GAL4/UAS system used in the fly, we have made progress developing a tool for mutating and revealing the expression of mouse genes by generating the tTA transactivator in the presence of a secondary TRE-regulated reporter molecule. A vector like the gene
Full Text Available In addition to genetic variation, epigenetic variation and transposons can greatly affect the evolutionary fitnesses landscape and gene expression. Previously we proposed a mathematical treatment of a general epigenetic variation model that we called Stochastic Epigenetic Modification (SEM model. In this study we follow up with a special case, the Transposon Silencing Model (TSM, with, once again, emphasis on quantitative treatment. We have investigated the evolutionary effects of epigenetic changes due to transposon (T insertions; in particular, we have considered a typical gene locus A and postulated that (i the expression level of gene A depends on the epigenetic state (active or inactive of a cis- located transposon element T, (ii stochastic variability in the epigenetic silencing of T occurs only in a short window of opportunity during development, (iii the epigenetic state is then stable during further development, and (iv the epigenetic memory is fully reset at each generation. We develop the model using two complementary approaches: a standard analytical population genetics framework (di usion equations and Monte-Carlo simulations. Both approaches led to similar estimates for the probability of fixation and time of fixation of locus TA with initial frequency P in a randomly mating diploid population of effective size Ne. We have ascertained the e ect that ρ, the probability of transposon Modification during the developmental window, has on the population (species. One of our principal conclusions is that as ρ increases, the pattern of fixation of the combined TA locus goes from "neutral" to "dominant" to "over-dominant". We observe that, under realistic values of ρ, epigenetic Modifications can provide an e cient mechanism for more rapid fixation of transposons and cis-located gene alleles. The results obtained suggest that epigenetic silencing, even if strictly transient (being reset at each generation, can still have signi cant
Yergeau Donald A
Full Text Available Abstract Background The Sleeping Beauty (SB transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization. Results Transgenic frogs that express the SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'. Conclusions In this study, we demonstrate that SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.
Revised genomic consensus for the hypermethylated CpG island region of the human L1 transposon and integration sites of full length L1 elements from recombinant clones made using methylation-tolerant host strains
Crowther, P J; Doherty, J P; Linsenmeyer, M E
preferentially from L1 members which have accumulated mutations that have removed sites of methylation. We present a revised consensus from the 5' presumptive control region of these elements. This revised consensus contains a consensus RNA polymerase III promoter which would permit the synthesis of transcripts......Efficient recovery of clones from the 5' end of the human L1 dispersed repetitive elements necessitates the use of deletion mcr- host strains since this region contains a CpG island which is hypermethylated in vivo. Clones recovered with conventional mcr+ hosts seem to have been derived...... from the 5' end of full length L1 elements. Such potential transcripts are likely to exhibit a high degree of secondary structure. In addition, we have determined the flanking sequences for 6 full length L1 elements. The majority of full length L1 clones show no convincing evidence for target site...
Full Text Available Abstract Background Bacterial artificial chromosomes (BACs are among the most widely used tools for studies of gene regulation and function in model vertebrates, yet methods for predictable delivery of BAC transgenes to the genome are currently limited. This is because BAC transgenes are usually microinjected as naked DNA into fertilized eggs and are known to integrate as multi-copy concatamers in the genome. Although conventional methods for BAC transgenesis have been very fruitful, complementary methods for generating single copy BAC integrations would be desirable for many applications. Results We took advantage of the precise cut-and-paste behavior of a natural transposon, Tol2, to develop a new method for BAC transgenesis. In this new method, the minimal sequences of the Tol2 transposon were used to deliver precisely single copies of a ~70 kb BAC transgene to the zebrafish and mouse genomes. We mapped the BAC insertion sites in the genome by standard PCR methods and confirmed transposase-mediated integrations. Conclusion The Tol2 transposon has a surprisingly large cargo capacity that can be harnessed for BAC transgenesis. The precise delivery of single-copy BAC transgenes by Tol2 represents a useful complement to conventional BAC transgenesis, and could aid greatly in the production of transgenic fish and mice for genomics projects, especially those in which single-copy integrations are desired.
Kumar, Rajesh; Grover, Sunita; Kaushik, Jai K; Batish, Virender Kumar
Lactobacillus plantarum is a flexible and versatile microorganism that inhabits a variety of niches, and its genome may express up to four bsh genes to maximize its survival in the mammalian gut. However, the ecological significance of multiple bsh genes in L. plantarum is still not clearly understood. Hence, this study demonstrated the disruption of bile salt hydrolase (bsh1) gene due to the insertion of a transposable element in L. plantarum Lp20 - a wild strain of human fecal origin. Surprisingly, L. plantarum strain Lp20 produced a ∼2.0 kb bsh1 amplicon against the normal size (∼1.0 kb) bsh1 amplicon of Bsh(+)L. plantarum Lp21. Strain Lp20 exhibited minimal Bsh activity in spite of having intact bsh2, bsh3 and bsh4 genes in its genome and hence had a Bsh(-) phenotype. Cloning and sequence characterization of Lp20 bsh1 gene predicted four individual open reading frames (ORFs) within this region. BLAST analysis of ORF1 and ORF2 revealed significant sequence similarity to the L. plantarum bsh1 gene while ORF3 and ORF4 showed high sequence homology to IS30-family transposases. Since, IS30-related transposon element was inserted within Lp20 bsh1 gene in reverse orientation (3'-5'), it introduced several stop codons and disrupted the protein reading frames of both Bsh1 and transposase. Inverted terminal repeats (GGCAGATTG) of transposon, mediated its insertion at 255-263 nt and 1301-1309 nt positions of Lp20 bsh1 gene. In conclusion, insertion of IS30 related-transposon within the bsh1 gene sequence of L. plantarum strain Lp20 demolished the integrity and functionality of Bsh1 enzyme. Additionally, this transposon DNA sequence remains active among various Lactobacillus spp. and hence harbors the potential to be explored in the development of efficient insertion mutagenesis system. Copyright © 2013 Elsevier GmbH. All rights reserved.
Bychenko, O S; Sukhanova, L V; Azhikina, T L; Sverdlov, E D
Two representatives of Baikal ciscoes - lake cisco and omul - diverged from a common ancestor as recently as 10-20 thousand years ago. We have found an increasing expression level of DTSsa4 Tc1-like DNA transposons in cisco and omul brains. The mapping of the sequences of these transposons from Salmo salar and Danio rerio genomes has shown that in some cases, these transposons are located in the 5' and 3' regions, as well as in the promoter regions of various genes. Probably, Tc1-like transposons affect the activity of neighboring genes, providing the adaptive divergence of the cisco population.
Urbach, Jonathan M; Wei, Tao; Liberati, Nicole; Grenfell-Lee, Daniel; Villanueva, Jacinto; Wu, Gang; Ausubel, Frederick M
PATIMDB is a software package for facilitating the generation of transposon mutant insertion libraries. The software has two main functions: process tracking and automated sequence analysis. The process tracking function specifically includes recording the status and fates of multiwell plates and samples in various stages of library construction. Automated sequence analysis refers specifically to the pipeline of sequence analysis starting with ABI files from a sequencing facility and ending with insertion location identifications. The protocols in this unit describe installation and use of PATIMDB software.
Full Text Available Abstract Background Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome. Results We performed a large-scale enhancer trap (ET screen and generated 338 insertions of the Tol2 transposon-based ET cassette into the zebrafish genome. These insertions were generated by remobilizing the transposon from two different donor sites in two transgenic lines. We found that 39% of Tol2 insertions occurred in transcription units, mostly into introns. Analysis of the transposon target sites revealed no strict specificity at the DNA sequence level. However, Tol2 was prone to target AT-rich regions with weak palindromic consensus sequences centered at the insertion site. Conclusion Our systematic analysis of sequential remobilizations of the Tol2 transposon from two independent sites within a vertebrate genome has revealed properties such as a tendency to integrate into transcription units and into AT-rich palindrome-like sequences. This information will influence the development of various applications involving DNA transposons and Tol2 in particular.
Kondrychyn, Igor; Garcia-Lecea, Marta; Emelyanov, Alexander; Parinov, Sergey; Korzh, Vladimir
Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome. We performed a large-scale enhancer trap (ET) screen and generated 338 insertions of the Tol2 transposon-based ET cassette into the zebrafish genome. These insertions were generated by remobilizing the transposon from two different donor sites in two transgenic lines. We found that 39% of Tol2 insertions occurred in transcription units, mostly into introns. Analysis of the transposon target sites revealed no strict specificity at the DNA sequence level. However, Tol2 was prone to target AT-rich regions with weak palindromic consensus sequences centered at the insertion site. Our systematic analysis of sequential remobilizations of the Tol2 transposon from two independent sites within a vertebrate genome has revealed properties such as a tendency to integrate into transcription units and into AT-rich palindrome-like sequences. This information will influence the development of various applications involving DNA transposons and Tol2 in particular.
Weber, J Mark; Reeves, Andrew; Cernota, William H; Wesley, Roy K
Transposon mutagenesis is an invaluable technique in molecular biology for the creation of random mutations that can be easily identified and mapped. However, in the field of microbial strain improvement, transposon mutagenesis has scarcely been used; instead, chemical and physical mutagenic methods have been traditionally favored. Transposons have the advantage of creating single mutations in the genome, making phenotype to genotype assignments less challenging than with traditional mutagens which commonly create multiple mutations in the genome. The site of a transposon mutation can also be readily mapped using DNA sequencing primer sites engineered into the transposon termini. In this chapter an in vitro method for transposon mutagenesis of Saccharopolyspora erythraea is presented. Since in vivo transposon tools are not available for most actinomycetes including S. erythraea, an in vitro method was developed. The in vitro method involves a significant investment in time and effort to create the mutants, but once the mutants are made and screened, a large number of highly relevant mutations of direct interest to erythromycin production can be found.
Qingshu Meng; Kaifu Chen; Lina Ma; Songnian Hu; Jun Yu
Transposons are sequence elements widely distributed among genomes of all three kingdoms of life, providing genomic changes and playing significant roles in genome evolution. Trichomonas vaginalis is an excellent model system for transposon study since its genome ( ～ 160 Mb) has been sequenced and is composed of ～65% transposons and other repetitive elements. In this study, we primarily report the identification of Kolobok-type transposons (termed tvBac) in T. vaginalis and the results of transposase sequence analysis. We categorized 24 novel subfamilies of the Kolobok element, including one autonomous subfamily and 23 non-autonomous subfamilies. We also identified a novel H2CH motif in tvBac transposases based on multiple sequence alignment. In addition, we supposed that tvBac and Mutator transposons may have evolved independently from a common ancestor according to our phylogenetic analysis. Our results provide basic information for the understanding of the function and evolution of tvBac transposons in particular and other related transposon families in general.
Takeda, Haruna; Rust, Alistair G; Ward, Jerrold M; Yew, Christopher Chin Kuan; Jenkins, Nancy A; Copeland, Neal G
Mutations in SMAD4 predispose to the development of gastrointestinal cancer, which is the third leading cause of cancer-related deaths. To identify genes driving gastric cancer (GC) development, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in the stomach of Smad4(+/-) mutant mice. This screen identified 59 candidate GC trunk drivers and a much larger number of candidate GC progression genes. Strikingly, 22 SB-identified trunk drivers are known or candidate cancer genes, whereas four SB-identified trunk drivers, including PTEN, SMAD4, RNF43, and NF1, are known human GC trunk drivers. Similar to human GC, pathway analyses identified WNT, TGF-β, and PI3K-PTEN signaling, ubiquitin-mediated proteolysis, adherens junctions, and RNA degradation in addition to genes involved in chromatin modification and organization as highly deregulated pathways in GC. Comparative oncogenomic filtering of the complete list of SB-identified genes showed that they are highly enriched for genes mutated in human GC and identified many candidate human GC genes. Finally, by comparing our complete list of SB-identified genes against the list of mutated genes identified in five large-scale human GC sequencing studies, we identified LDL receptor-related protein 1B (LRP1B) as a previously unidentified human candidate GC tumor suppressor gene. In LRP1B, 129 mutations were found in 462 human GC samples sequenced, and LRP1B is one of the top 10 most deleted genes identified in a panel of 3,312 human cancers. SB mutagenesis has, thus, helped to catalog the cooperative molecular mechanisms driving SMAD4-induced GC growth and discover genes with potential clinical importance in human GC.
Desai, Prerak; Porwollik, Steffen; Canals, Rocio; Perez, Daniel R.; Chu, Weiping; McClelland, Michael; Teplitski, Max
ABSTRACT Human enteric pathogens, such as Salmonella spp. and verotoxigenic Escherichia coli, are increasingly recognized as causes of gastroenteritis outbreaks associated with the consumption of fruits and vegetables. Persistence in plants represents an important part of the life cycle of these pathogens. The identification of the full complement of Salmonella genes involved in the colonization of the model plant (tomato) was carried out using transposon insertion sequencing analysis. With this approach, 230,000 transposon insertions were screened in tomato pericarps to identify loci with reduction in fitness, followed by validation of the screen results using competition assays of the isogenic mutants against the wild type. A comparison with studies in animals revealed a distinct plant-associated set of genes, which only partially overlaps with the genes required to elicit disease in animals. De novo biosynthesis of amino acids was critical to persistence within tomatoes, while amino acid scavenging was prevalent in animal infections. Fitness reduction of the Salmonella amino acid synthesis mutants was generally more severe in the tomato rin mutant, which hyperaccumulates certain amino acids, suggesting that these nutrients remain unavailable to Salmonella spp. within plants. Salmonella lipopolysaccharide (LPS) was required for persistence in both animals and plants, exemplifying some shared pathogenesis-related mechanisms in animal and plant hosts. Similarly to phytopathogens, Salmonella spp. required biosynthesis of amino acids, LPS, and nucleotides to colonize tomatoes. Overall, however, it appears that while Salmonella shares some strategies with phytopathogens and taps into its animal virulence-related functions, colonization of tomatoes represents a distinct strategy, highlighting this pathogen's flexible metabolism. IMPORTANCE Outbreaks of gastroenteritis caused by human pathogens have been increasingly associated with foods of plant origin, with tomatoes
Hu, Bingjie; Zhou, Mingbing
Miniature inverted-repeat transposable elements transposon is a special transposon that could transpose by "cut-paste" mechanism, which is one of characteristics of DNA transposons. Otherwise, the copy number of MITEs is very high, which is one of characteristics of RNA transposons. Many MITE families have been reported, but little about active MITEs. We summarize recent advances in studying active MITEs. Most the MITEs belong to the Tourist-like family, such as mPing, mGing, PhTourist1, Tmi1 and PhTst-3. Additionally, DTstu1 and MITE-39 belong to Stowaway-like family, and AhMITEs1 belongs to Mutator-like family. Moreover, we summarize the structure (terminal inverse repeats and target site duplications), copy number, evolution pattern and transposition characteristics of these active MITEs, to provide the foundation for the identification of other active MITEs and subsequent research on MITE transposition and amplification mechanism.
Kirchner, O; Gartemann, K H; Zellermann, E M; Eichenlaub, R; Burger, A
A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 x 10(6) transformants per microg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 x 10(3) transposon mutants per pg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.
de Ruiter, Julian R; Kas, Sjors M; Schut, Eva; Adams, David J; Koudijs, Marco J; Wessels, Lodewyk F A; Jonkers, Jos
Insertional mutagenesis using engineered transposons is a potent forward genetic screening technique used to identify cancer genes in mouse model systems. In the analysis of these screens, transposon insertion sites are typically identified by targeted DNA-sequencing and subsequently assigned to predicted target genes using heuristics. As such, these approaches provide no direct evidence that insertions actually affect their predicted targets or how transcripts of these genes are affected. To address this, we developed IM-Fusion, an approach that identifies insertion sites from gene-transposon fusions in standard single- and paired-end RNA-sequencing data. We demonstrate IM-Fusion on two separate transposon screens of 123 mammary tumors and 20 B-cell acute lymphoblastic leukemias, respectively. We show that IM-Fusion accurately identifies transposon insertions and their true target genes. Furthermore, by combining the identified insertion sites with expression quantification, we show that we can determine the effect of a transposon insertion on its target gene(s) and prioritize insertions that have a significant effect on expression. We expect that IM-Fusion will significantly enhance the accuracy of cancer gene discovery in forward genetic screens and provide initial insight into the biological effects of insertions on candidate cancer genes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
Muszewska, Anna; Steczkiewicz, Kamil; Stepniewska-Dziubinska, Marta; Ginalski, Krzysztof
Transposable elements (TEs) shape genomes via recombination and transposition, lead to chromosomal rearrangements, create new gene neighborhoods, and alter gene expression. They play key roles in adaptation either to symbiosis in Amanita genus or to pathogenicity in Pyrenophora tritici-repentis. Despite growing evidence of their importance, the abundance and distribution of mobile elements replicating in a "cut-and-paste" fashion is barely described so far. In order to improve our knowledge on this old and ubiquitous class of transposable elements, 1,730 fungal genomes were scanned using both de novo and homology-based approaches. DNA TEs have been identified across the whole data set and display uneven distribution from both DNA TE classification and fungal taxonomy perspectives. DNA TE content correlates with genome size, which confirms that many transposon families proliferate simultaneously. In contrast, it is independent from intron density, average gene distance and GC content. TE count is associated with species' lifestyle and tends to be elevated in plant symbionts and decreased in animal parasites. Lastly, we found that fungi with both RIP and RNAi systems have more total DNA TE sequences but less elements retaining a functional transposase, what reflects stringent control over transposition. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Oliveira Sarah G
Full Text Available Abstract Background Active transposable elements (TEs can be passed between genomes of different species by horizontal transfer (HT. This may help them to avoid vertical extinction due to elimination by natural selection or silencing. HT is relatively frequent within eukaryotic taxa, but rare between distant species. Findings Closely related Mariner-type DNA transposon families, collectively named as Mariner-1_Tbel families, are present in the genomes of two ants and two mammalian genomes. Consensus sequences of the four families show pairwise identities greater than 95%. In addition, mammalian Mariner1_BT family shows a close evolutionary relationship with some insect Mariner families. Mammalian Mariner1_BT type sequences are present only in species from three groups including ruminants, tooth whales (Odontoceti, and New World leaf-nosed bats (Phyllostomidae. Conclusions Horizontal transfer accounts for the presence of Mariner_Tbel and Mariner1_BT families in mammals. Mariner_Tbel family was introduced into hedgehog and tree shrew genomes approximately 100 to 69 million years ago (MYA. Most likely, these TE families were transferred from insects to mammals, but details of the transfer remain unknown.
Oliveira, Sarah G; Bao, Weidong; Martins, Cesar; Jurka, Jerzy
Active transposable elements (TEs) can be passed between genomes of different species by horizontal transfer (HT). This may help them to avoid vertical extinction due to elimination by natural selection or silencing. HT is relatively frequent within eukaryotic taxa, but rare between distant species. Closely related Mariner-type DNA transposon families, collectively named as Mariner-1_Tbel families, are present in the genomes of two ants and two mammalian genomes. Consensus sequences of the four families show pairwise identities greater than 95%. In addition, mammalian Mariner1_BT family shows a close evolutionary relationship with some insect Mariner families. Mammalian Mariner1_BT type sequences are present only in species from three groups including ruminants, tooth whales (Odontoceti), and New World leaf-nosed bats (Phyllostomidae). Horizontal transfer accounts for the presence of Mariner_Tbel and Mariner1_BT families in mammals. Mariner_Tbel family was introduced into hedgehog and tree shrew genomes approximately 100 to 69 million years ago (MYA). Most likely, these TE families were transferred from insects to mammals, but details of the transfer remain unknown.
Balter, H.; Griffith, C.S.; American Museum of Natural History, New York; Margulies, L.
The interaction of X-ray-induced and transposon-induced damage was investigated in P-M hybrid dysgenesis in Drosophila melanogaster. X-ray dose-response of 330-1320 rad was monitored for sterility, fecundicity and partial X/Y chromosome loss among F 2 progeny derived from dysgenic cross of M strain females xP strain males (cross A) and its reciprocal (cross B), using a weaker and the standard Harwich P strain subline. The synergistic effect of P element activity and X-rays on sterility was observed only in cross A hybrids and the dose-response was nonlinear in hybrids derived from the strong standard reference Harwich subline, H W . This finding suggests that lesions induced by both mutator systems which produce the synergistic effects are 2-break events. Effect of increasing dose on the decline of fecundicity was synergistic, but linear, in hybrids of either subline. There was no interaction evident and thus no synergism in X/Y nondisjunction and partial Y chromosome loss measured by the loss of the B s marker alone or together with the y + marker. Interaction was detected in the loss of the y + marker alone from the X and Y chromosomes. The possible three-way interaction of X-rays (660 rad), post-replication repair deficiency and P elements mobility was assessed by measuring transmission distortion in dysgenic males derived from the Π 2 P strain. (author). 38 refs.; 5 tabs
Katayama, Masafumi; Hirayama, Takashi; Tani, Tetsuya; Nishimori, Katsuhiko; Onuma, Manabu; Fukuda, Tomokazu
Induced pluripotent stem (iPS) cell technology lead terminally differentiated cells into the pluripotent stem cells through the expression of defined reprogramming factors. Although, iPS cells have been established in a number of mammalian species, including mouse, human, and monkey, studies on iPS cells in avian species are still very limited. To establish chick iPS cells, six factors were used within the poly-cistronic reprogramming vector (PB-R6F), containing M3O (MyoD derived transactivation domain fused with Oct3/4), Sox2, Klf4, c-Myc, Lin28, and Nanog. The PB-R6F derived iPS cells were alkaline-phosphatase and SSEA-1 positive, which are markers of pluripotency. Elevated levels of endogenous Oct3/4 and Nanog genes were detected in the established iPS cells, suggesting the activation of the FGF signaling pathway is critical for the pluripotent status. Histological analysis of teratoma revealed that the established chick iPS cells have differentiation ability into three-germ-layer derived tissues. This is the first report of establishment of avian derived iPS cells with a single poly-cistronic transposon based expression system. The establishment of avian derived iPS cells could contribute to the genetic conservation and modification of avian species. © 2017 Wiley Periodicals, Inc.
Wong, Yee-Chin; Pain, Arnab; Nathan, Sheila
Burkholderia cenocepacia, a pathogenic member of the Burkholderia cepacia complex (Bcc), has emerged as a significant threat towards cystic fibrosis patients, where infection often leads to the fatal clinical manifestation known as cepacia syndrome. Many studies have investigated the pathogenicity of B. cenocepacia as well as its ability to become highly resistant towards many of the antibiotics currently in use. In addition, studies have also been undertaken to understand the pathogen's capacity to adapt and survive in a broad range of environments. Transposon based mutagenesis has been widely used in creating insertional knock-out mutants and coupled with recent advances in sequencing technology, robust tools to study gene function in a genome-wide manner have been developed based on the assembly of saturated transposon mutant libraries. In this study, we describe the construction of a large-scale library of B. cenocepacia transposon mutants. To create transposon mutants of B. cenocepacia strain J2315, electrocompetent bacteria were electrotransformed with the EZ-Tn5 transposome. Tetracyline resistant colonies were harvested off selective agar and pooled. Mutants were generated in multiple batches with each batch consisting of ˜20,000 to 40,000 mutants. Transposon insertion was validated by PCR amplification of the transposon region. In conclusion, a saturated B. cenocepacia J2315 transposon mutant library with an estimated total number of 500,000 mutants was successfully constructed. This mutant library can now be further exploited as a genetic tool to assess the function of every gene in the genome, facilitating the discovery of genes important for bacterial survival and adaptation, as well as virulence.
Dwibedi, Chinmay Kumar; Sjöström, Karin; Edquist, Petra; Wai, Sun Nyunt; Uhlin, Bernt Eric
Acinetobacter baumannii has emerged as an important opportunistic pathogen equipped with a growing number of antibiotic resistance genes. Our study investigated the molecular epidemiology and antibiotic resistance features of 28 consecutive carbapenem-resistant clinical isolates of A. baumannii collected throughout Sweden in 2012 and 2013. The isolates mainly belonged to clonal complexes (CCs) with an extensive international distribution, such as CC2 (n = 16) and CC25 (n = 7). Resistance to carbapenems was related to blaOXA-23 (20 isolates), blaOXA-24/40-like (6 isolates), blaOXA-467 (1 isolate), and ISAba1-blaOXA-69 (1 isolate). Ceftazidime resistance was associated with blaPER-7 in the CC25 isolates. Two classical point mutations were responsible for resistance to quinolones in all the isolates. Isolates with high levels of resistance to aminoglycosides carried the 16S rRNA methylase armA gene. The isolates also carried a variety of genes encoding aminoglycoside-modifying enzymes. Several novel structures involved in aminoglycoside resistance were identified, including Tn6279, ΔTn6279, Ab-ST3-aadB, and different assemblies of Tn6020 and TnaphA6. Importantly, a number of circular forms related to the IS26 or ISAba125 composite transposons were detected. The frequent occurrence of these circular forms in the populations of several isolates indicates a potential role of these circular forms in the dissemination of antibiotic resistance genes. PMID:26824943
Davidson William S
Full Text Available Abstract Background Several genome duplications have occurred in the evolutionary history of teleost fish. In returning to a stable diploid state, the polyploid genome reorganized, and large portions are lost, while the fish lines evolved to numerous species. Large scale transposon movement has been postulated to play an important role in the genome reorganization process. We analyzed the DNA sequence of several large loci in Salmo salar and other species for the presence of DNA transposon families. Results We have identified bursts of activity of 14 families of DNA transposons (12 Tc1-like and 2 piggyBac-like families, including 11 novel ones in genome sequences of Salmo salar. Several of these families have similar sequences in a number of closely and distantly related fish, lamprey, and frog species as well as in the parasite Schistosoma japonicum. Analysis of sequence similarities between copies within the families of these bursts demonstrates several waves of transposition activities coinciding with salmonid species divergence. Tc1-like families show a master gene-like copying process, illustrated by extensive but short burst of copying activity, while the piggyBac-like families show a more random copying pattern. Recent families may include copies with an open reading frame for an active transposase enzyme. Conclusion We have identified defined bursts of transposon activity that make use of master-slave and random mechanisms. The bursts occur well after hypothesized polyploidy events and coincide with speciation events. Parasite-mediated lateral transfer of transposons are implicated.
Full Text Available Transposon mutagenesis, in combination with parallel sequencing, is becoming a powerful tool for en-masse mutant analysis. A probability generating function was used to explain observed miniHimar transposon insertion patterns, and gene essentiality calls were made by transposon insertion frequency analysis (TIFA. TIFA incorporated the observed genome and sequence motif bias of the miniHimar transposon. The gene essentiality calls were compared to: 1 previous genome-wide direct gene-essentiality assignments; and, 2 flux balance analysis (FBA predictions from an existing genome-scale metabolic model of Shewanella oneidensis MR-1. A three-way comparison between FBA, TIFA, and the direct essentiality calls was made to validate the TIFA approach. The refinement in the interpretation of observed transposon insertions demonstrated that genes without insertions are not necessarily essential, and that genes that contain insertions are not always nonessential. The TIFA calls were in reasonable agreement with direct essentiality calls for S. oneidensis, but agreed more closely with E. coli essentiality calls for orthologs. The TIFA gene essentiality calls were in good agreement with the MR-1 FBA essentiality predictions, and the agreement between TIFA and FBA predictions was substantially better than between the FBA and the direct gene essentiality predictions.
Full Text Available Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.
Paun, Linda; Nitsche, Benjamin; Homan, Tim; Ram, Arthur F; Kempken, Frank
The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background.
Greco, R.; Ouwerkerk, P.B.F.; Taal, A.J.C.; Sallaud, C.; Guiderdoni, E.; Meijer, A.H.; Hoge, J.H.C.; Pereira, A.B.
Transposition of the maize En/Spm system in rice was investigated using a two-component construct consisting of an immobilised transposase source driven by the CaMV 35S-promoter, and a modified I/dSpm transposon. Mobilization of I/dSpm in somatic sectors was demonstrated by sequencing of excision
Yokoo, Masako; Fujita, Ryosuke; Nakajima, Yumiko; Yoshimizu, Mamoru; Kasai, Hisae; Asano, Shin-ichiro; Bando, Hisanori
Highlights: •The baculovirus vector infiltrates the cells of economic important fishes. •Drosophila Mos1 transposase expressed in fish cells maintains its ability to localize to the nucleus. •The baculoviral vector carrying Mos1 is a useful tool to stably transform fish cells. -- Abstract: Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells
Yokoo, Masako [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Fujita, Ryosuke [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Innate Immunity Laboratory, Graduate School of Life Science and Creative Research Institution, Hokkaido University, Sapporo 001-0021 (Japan); Nakajima, Yumiko [Functional Genomics Group, COMB, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa 903-0213 (Japan); Yoshimizu, Mamoru; Kasai, Hisae [Faculty of Fisheries Sciences, Hokkaido University, Hakodate 041-8611 (Japan); Asano, Shin-ichiro [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Bando, Hisanori, E-mail: email@example.com [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan)
Highlights: •The baculovirus vector infiltrates the cells of economic important fishes. •Drosophila Mos1 transposase expressed in fish cells maintains its ability to localize to the nucleus. •The baculoviral vector carrying Mos1 is a useful tool to stably transform fish cells. -- Abstract: Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.
Burgis Timothy A
Full Text Available Abstract Background In recent years there has been an increasing problem with Staphylococcus aureus strains that are resistant to treatment with existing antibiotics. An important starting point for the development of new antimicrobial drugs is the identification of "essential" genes that are important for bacterial survival and growth. Results We have developed a robust microarray and PCR-based method, Transposon-Mediated Differential Hybridisation (TMDH, that uses novel bioinformatics to identify transposon inserts in genome-wide libraries. Following a microarray-based screen, genes lacking transposon inserts are re-tested using a PCR and sequencing-based approach. We carried out a TMDH analysis of the S. aureus genome using a large random mariner transposon library of around a million mutants, and identified a total of 351 S. aureus genes important for survival and growth in culture. A comparison with the essential gene list experimentally derived for Bacillus subtilis highlighted interesting differences in both pathways and individual genes. Conclusion We have determined the first comprehensive list of S. aureus essential genes. This should act as a useful starting point for the identification of potential targets for novel antimicrobial compounds. The TMDH methodology we have developed is generic and could be applied to identify essential genes in other bacterial pathogens.
Maglennon, Gareth A; Cook, Beth S; Deeney, Alannah S; Bossé, Janine T; Peters, Sarah E; Langford, Paul R; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N
Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.
Full Text Available Abstract Background The molecular organization of very few genetically defined CACTA transposon systems have been characterized thoroughly as those of Spm/En in maize, Tam1 of Antirrhinum majus Candystripe1 (Cs1 from Sorghum bicolor and CAC1 from Arabidopsis thaliana, for example. To date, only defective deletion derivatives of CACTA elements have been described for soybean, an economically important plant species whose genome sequence will be completed in 2008. Results We identified a 20.5 kb insertion in a soybean flavonoid 3'-hydroxylase (F3'H gene representing the t* allele (stable gray trichome color whose origin traces to a single mutable chimeric plant displaying both tawny and gray trichomes. This 20.5 kb insertion has the molecular structure of a putative autonomous transposon of the CACTA family, designated Tgmt*. It encodes a large gene that was expressed in two sister isolines (T* and tm of the stable gray line (t* from which Tgmt* was isolated. RT-PCR derived cDNAs uncovered the structure of a large precursor mRNA as well as alternatively spliced transcripts reminiscent of the TNPA-mRNA generated by the En-1 element of maize but without sequence similarity to the maize TNPA. The larger mRNA encodes a transposase with a tnp2 and TNP1-transposase family domains. Because the two soybean lines expressing Tgmt* were derived from the same mutable chimeric plant that created the stable gray trichome t* allele line from which the element was isolated, Tgmt* has the potential to be an autonomous element that was rapidly inactivated in the stable gray trichome t* line. Comparison of Tgmt* to previously described Tgm elements demonstrated that two subtypes of CACTA transposon families exist in soybean based on divergence of their characteristic subterminal repeated motifs and their transposases. In addition, we report the sequence and annotation of a BAC clone containing the F3'H gene (T locus which was interrupted by the novel Tgmt* element
Full Text Available Transposon insertion sequencing (TIS; also known as TnSeq is a potent approach commonly used to comprehensively define the genetic loci that contribute to bacterial fitness in diverse environments. A key presumption underlying analyses of TIS datasets is that loci with a low frequency of transposon insertions contribute to fitness. However, it is not known whether factors such as nucleoid binding proteins can alter the frequency of transposon insertion and thus whether TIS output may systematically reflect factors that are independent of the role of the loci in fitness. Here, we investigated whether the histone-like nucleoid structuring (H-NS protein, which preferentially associates with AT-rich sequences, modulates the frequency of Mariner transposon insertion in the Vibrio cholerae genome, using comparative analysis of TIS results from wild-type (wt and Δhns V. cholerae strains. These analyses were overlaid on gene classification based on GC content as well as on extant genome-wide identification of H-NS binding loci. Our analyses revealed a significant dearth of insertions within AT-rich loci in wt V. cholerae that was not apparent in the Δhns insertion library. Additionally, we observed a striking correlation between genetic loci that are overrepresented in the Δhns insertion library relative to their insertion frequency in wt V. cholerae and loci previously found to physically interact with H-NS. Collectively, our findings reveal that factors other than genetic fitness can systematically modulate the frequency of transposon insertions in TIS studies and add a cautionary note to interpretation of TIS data, particularly for AT-rich sequences.
Filée, Jonathan; Rouault, Jacques-Deric; Harry, Myriam; Hua-Van, Aurélie
The Triatomine bug Rhodnius prolixus is a vector of Trypanosoma cruzi, which causes the Chagas disease in Latin America. R. prolixus can also transfer transposable elements horizontally across a wide range of species. We have taken advantage of the availability of the 700 Mbp complete genome sequence of R. prolixus to study the dynamics of invasion and persistence of transposable elements in this species. Using both library-based and de novo methods of transposon detection, we found less than 6 % of transposable elements in the R. prolixus genome, a relatively low percentage compared to other insect genomes with a similar genome size. DNA transposons are surprisingly abundant and elements belonging to the mariner family are by far the most preponderant components of the mobile part of this genome with 11,015 mariner transposons that could be clustered in 89 groups (75 % of the mobilome). Our analysis allowed the detection of a new mariner clade in the R. prolixus genome, that we called nosferatis. We demonstrated that a large diversity of mariner elements invaded the genome and expanded successfully over time via three main processes. (i) several families experienced recent and massive expansion, for example an explosive burst of a single mariner family led to the generation of more than 8000 copies. These recent expansion events explain the unusual prevalence of mariner transposons in the R. prolixus genome. Other families expanded via older bursts of transposition demonstrating the long lasting permissibility of mariner transposons in the R. prolixus genome. (ii) Many non-autonomous families generated by internal deletions were also identified. Interestingly, two non autonomous families were generated by atypical recombinations (5' part replacement with 3' part). (iii) at least 10 cases of horizontal transfers were found, supporting the idea that host/vector relationships played a pivotal role in the transmission and subsequent persistence of transposable
Rella, M; Axelrood, P E; Weinhold, A R; Schroth, M N
The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled transposon mutagenesis of a wild-type antibiotic-producing strain of E. carotovora subsp. betavasculorum. Twelve antibiotic-negative mutants were isolated, and one of these showed a reduction in antibiotic production in vitro. Many of these mutants also showed a reduction in their ability to macerate potato tissue. The mutants were classified into four genetic groups on the basis of their genetic and phenotypic characteristics, indicating that several genes are involved in antibiotic biosynthesis by E. carotovora subsp. betavasculorum. PMID:2543291
Kues, Wilfried A.; Herrmann, Doris; Barg-Kues, Brigitte
the nonviral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4, and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed induced pluripotent stem (i......PS) cells showed long-term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5, and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives......The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing...
Tabara, H; Sarkissian, M; Kelly, W G; Fleenor, J; Grishok, A; Timmons, L; Fire, A; Mello, C C
Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.
Fedashchin, Andrij; Cernota, William H.; Gonzalez, Melissa C.; Leach, Benjamin I.; Kwan, Noelle; Wesley, Roy K.; Weber, J. Mark
A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ∼3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering. PMID:26468041
Rella, M; Axelrood, P E; Weinhold, A R; Schroth, M N
The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled tra...
Full Text Available Transposable elements are capable of inducing heritable de novo genetic variation. The sequences capable of reactivation, and environmental factors that induce mobilization, remain poorly defined even in well-studied genomes such as maize. We treated maize tissue culture with the demethylating agent 5-aza-2-deoxcytidine and examined long-term tissue culture lines to discover silenced transposable elements that have the potential to induce heritable genetic variation. Through these screens we have identified a novel low copy number hAT transposon, Tissue Culture Up-Regulated (TCUP, which is transcribed at high levels in long-term maize Black Mexican Sweet (BMS tissue culture and up-regulated in response to treatment with 5-aza-2-deoxycytidine. Analysis of the TIGR Maize Gene Index revealed that this element is the most frequently represented EST from the BMS cell culture library and is not represented in other tissue libraries, which is the basis for its name. A full-length sequence was assembled in inbred B73 that contains the putative functional motifs required for autonomous movement of a hAT transposon. Transposon display detected movement of TCUP in two long-term tissue cultured cell lines of the genotype Hi-II AxB and BMS. This research implicates TCUP as a transposon that is capable of reactivation and which may also be particularly sensitive to the stress of the tissue culture environment. Our findings are consistent with the hypothesis that epigenetic alterations potentiate genomic responses to stress during clonal propagation of plants.
Full Text Available Abstract Background Gene knockouts are a critical resource for functional genomics. In Arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic DNA flanking insertion mutants. These Flanking Sequence Tags (FSTs map each mutant to a specific locus within the genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also generate somatic insertions that fail to segregate in subsequent generations. Results Transposon insertion sites from 106 UniformMu FSTs were tested for inheritance by locus-specific PCR. We confirmed 89% of the FSTs to be germinal transposon insertions. We found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. Instead, this subset of insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic sequences. The locus-specific PCR assays identified a knockout of a 6-phosphogluconate dehydrogenase gene that co-segregates with a seed mutant phenotype. The mutant phenotype linked to this knockout generates novel hypotheses about the role for the plastid-localized oxidative pentose phosphate pathway during grain-fill. Conclusion We show that FSTs from the UniformMu population identify stable, germinal insertion sites in maize. Moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis. We conclude from these data that the current collection of 1,882 non-redundant insertion sites from UniformMu provide a genome-wide resource for reverse genetics.
Full Text Available Minimal piggyBac vectors are a modified single-plasmid version of the classical piggyBac delivery system that can be used for stable transgene integration. These vectors have a truncated terminal domain in the delivery cassette and thus, integrate significantly less flanking transposon DNA into host cell chromatin than classical piggyBac vectors. Herein, we test various characteristics of this modified transposon. The integration efficiency of minimal piggyBac vectors was inversely related to the size of both the transposon and the entire plasmid, but inserts as large as 15 kb were efficiently integrated. Open and super-coiled vectors demonstrated the same integration efficiency while DNA methylation decreased the integration efficiency and silenced the expression of previously integrated sequences in some cell types. Importantly, the incidence of plasmid backbone integration was not increased above that seen in nontransposon control vectors. In BALB/c mice, we demonstrated prolonged expression of two transgenes (intracellular mCherry and secretable Gaussia luciferase when delivered by the minimal piggyBac that resulted in a more sustained antibody production against the immunogenic luciferase than when delivered by a transient (nontransposon vector plasmid. We conclude that minimal piggyBac vectors are an effective alternative to other integrative systems for stable DNA delivery in vitro and in vivo.
Park, June-Shine; Kim, Bae-Hwan; Park, Sung Goo; Jung, Sun Young; Lee, Do Hee; Son, Woo-Chan
The Sleeping Beauty (SB) transposon system has been receiving much attention as a gene transfer method of choice since it allows permanent gene expression after insertion into the host chromosome. However, low transposition frequency in higher eukaryotes limits its use in commonly-used mammalian species. Researchers have therefore attempted to modify gene delivery and expression to overcome this limitation. In mouse liver, tumor induction using SB introduced by the hydrodynamic method has been successfully accomplished. Liver tumor in rat models using SB could also be of great use; however, dose of DNA, injection volume, rate of injection and achieving back pressure limit the use of the hydrodynamics-based gene delivery. In the present study, we combined the electroporation, a relatively simple and easy gene delivery method, with the SB transposon system and as a result successfully induced tumor in rat liver by directly injecting the c-Myc, HRAS and shp53 genes. The tumor phenotype was determined as a sarcomatoid carcinoma. To our knowledge, this is the first demonstration of induction of tumor in the rat liver using the electroporation-enhanced SB transposon system. Copyright © 2013 Elsevier Inc. All rights reserved.
Kubanejshvili, M.G.; Smirnov, S.P.; Tarasov, V.A.
Migration of ampicillin transposon Tn1 under normal conditions in Escherichia coli cells proceeds with low frequency (10 -4 transpositions for cell). The low transposition frequency is conditioned by the transposition repression, realized by the gene-repressor in transposon structure and, probably, by other regulating genes of the bacterium-host. E. coli cell treatment by physical and chemical mutagens resulted in induction of translocation of ampicillin transposon Tn1 from plasmid RP4 into other replicons. Mitomycin C and ultraviolet radiation produced stronger inducing effect as compared to nitroso-guanidine (NG). The effect of the given mutagens on transposition Tn1 correlated with their activating capacity with respect to inducible SOS-functions of E coli. The mutation of rec A didn't influence on spontaneous Tn1 transposition, but blocked completely the induction of transposition process under mutagen effect. The relationship of inducible transposition with SOS-functions in E. coli cells, controlled by recA and lexA genes, as well as the possible role of the process in genetic microorganism variability are discussed in the paper
Venken, Koen J T; Bellen, Hugo J
The study of genetics, genes, and chromosomal inheritance was initiated by Thomas Morgan in 1910, when the first visible mutations were identified in fruit flies. The field expanded upon the work initiated by Herman Muller in 1926 when he used X-rays to develop the first balancer chromosomes. Today, balancers are still invaluable to maintain mutations and transgenes but the arsenal of tools has expanded vastly and numerous new methods have been developed, many relying on the availability of the genome sequence and transposable elements. Forward genetic screens based on chemical mutagenesis or transposable elements have resulted in the unbiased identification of many novel players involved in processes probed by specific phenotypic assays. Reverse genetic approaches have relied on the availability of a carefully selected set of transposon insertions spread throughout the genome to allow the manipulation of the region in the vicinity of each insertion. Lastly, the ability to transform Drosophila with single copy transgenes using transposons or site-specific integration using the ΦC31 integrase has allowed numerous manipulations, including the ability to create and integrate genomic rescue constructs, generate duplications, RNAi knock-out technology, binary expression systems like the GAL4/UAS system as well as other methods. Here, we will discuss the most useful methodologies to interrogate the fruit fly genome in vivo focusing on chemical mutagenesis, transposons and transgenes. Genome engineering approaches based on nucleases and RNAi technology are discussed in following chapters. Copyright © 2014 Elsevier Inc. All rights reserved.
Full Text Available Mohammad R Alavi1,2, Vlado Antonic2, Adrien Ravizee1, Peter J Weina3, Mina Izadjoo1,2, Alexander Stojadinovic21Division of Wound Biology and Translational Research, Armed Forces Institute of Pathology and American Registry of Pathology, Washington DC, 2Combat Wound Initiative Program, Walter Reed Army Medical Center, Washington DC, 3The Walter Reed Army Institute of Research, Silver Spring, MD, USABackground: Genus Enterobacter includes important opportunistic nosocomial pathogens that could infect complex wounds. The presence of antibiotic resistance genes in these microorganisms represents a challenging clinical problem in the treatment of these wounds. In the authors’ screening of antibiotic-resistant bacteria from complex wounds, an Enterobacter species was isolated that harbors antibiotic-resistant plasmids conferring resistance to Escherichia coli. The aim of this study was to identify the resistance genes carried by one of these plasmids.Methods: The plasmids from the Enterobacter isolate were propagated in E. coli and one of the plasmids, designated as pR23, was sequenced by the Sanger method using fluorescent dye-terminator chemistry on a genetic analyzer. The assembled sequence was annotated by search of the GenBank database.Results: Plasmid pR23 is composed of the transposon Tn1331 and a backbone plasmid that is identical to the plasmid pPIGDM1 from Enterobacter agglomerans. The multidrug-resistance transposon Tn1331, which confers resistance to aminoglycoside and beta lactam antibiotics, has been previously isolated only from Klebsiella. The Enterobacter plasmid pPIGDM1, which carries a ColE1-like origin of replication and has no apparent selective marker, appears to provide a backbone for propagation of Tn1331 in Enterobacter. The recognition sequence of Tn1331 transposase for insertion into pPIGDM1 is the pentanucleotide TATTA, which occurs only once throughout the length of this plasmid.Conclusion: Transposition of Tn1331 into
Liu, Ying; Tan, Huiling; Tian, Hui; Liang, Chunyang; Chen, She; Liu, Qinghua
The effector of RNA interference (RNAi) is the RNA-induced silencing complex (RISC). C3PO promotes the activation of RISC by degrading the Argonaute2 (Ago2)-nicked passenger strand of duplex siRNA. Active RISC is a multiple-turnover enzyme that uses the guide strand of siRNA to direct the Ago2-mediated sequence-specific cleavage of complementary mRNA. How this effector step of RNAi is regulated is currently unknown. Here, we used the human Ago2 minimal RISC system to purify Sjögren's syndrome antigen B (SSB)/autoantigen La as an activator of the RISC-mediated mRNA cleavage activity. Our reconstitution studies showed that La could promote multiple-turnover RISC catalysis by facilitating the release of cleaved mRNA from RISC. Moreover, we demonstrated that La was required for efficient RNAi, antiviral defense, and transposon silencing in vivo. Taken together, the findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC. Copyright Â© 2011 Elsevier Inc. All rights reserved.
Mutations in the Histone-Like Nucleoid Structuring Regulatory Gene (hns) Decrease the Adherence of Shiga Toxin-Producing Escherichia coli 091:H21 Strain B2F1 to Human Colonic Epithelial Cells and Increase the Production of Hemolysin
...:H21 to human colonic epithelial cells. Transposon mutagenesis of B2F1 was accomplished with the mini-Tn5phoACm mobile element and a mutant bank of B2F1 colonies that carried putative in-frame PhoA-positive transposon insertions was isolated...
Hickman, Alison B; Ewis, Hosam E; Li, Xianghong; Knapp, Joshua A; Laver, Thomas; Doss, Anna-Louise; Tolun, Gökhan; Steven, Alasdair C; Grishaev, Alexander; Bax, Ad; Atkinson, Peter W; Craig, Nancy L; Dyda, Fred
Hermes is a member of the hAT transposon superfamily that has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. Although isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple nonspecific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA. Copyright © 2014 Elsevier Inc. All rights reserved.
Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K; Quach, Helen Ngoc Bao; Joshi, Adita; LaFave, Matthew C; Burgess, Shawn M; Sampath, Karuna
DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering. Copyright © 2016 Vrljicak et al.
Full Text Available Abstract Background Transposons constitute the major fractions of repetitive sequences in eukaryotes, and have been crucial in the shaping of current genomes. Transposons are generally divided into two classes according to the mechanism underlying their transposition: RNA intermediate class 1 and DNA intermediate class 2. CACTA is a class 2 transposon superfamily, which is found exclusively in plants. As some transposons, including the CACTA superfamily, are highly abundant in plant species, and their nucleotide sequences are highly conserved within a family, they can be utilized as genetic markers, using a slightly modified version of the conventional AFLP protocol. Rim2 /Hipa is a CACTA transposon family having 16 bp consensus TIR sequences to be present in high copy numbers in rice genome. This research was carried out in order to develop a Rim2/Hipa CACTA-AFLP or Rim2/Hipa CACTA-TD (transposon display, hereafter Rim2/Hipa-TD protocol for the study of genetic markers in map construction and the study of genetic diversity in rice. Results Rim2/Hipa-TD generated ample polymorphic profiles among the different rice accessions, and the amplification profiles were highly reproducible between different thermocyclers and Taq polymerases. These amplification profiles allowed for clear distinction between two different ecotypes, Japonica and Indica, of Oryza sativa. In the analysis of RIL populations, the Rim2/Hipa-TD markers were found to be segregated largely in a dominant manner, although in a few cases, non-parental bands were observed in the segregating populations. Upon linkage analysis, the Rim2/Hipa-TD markers were found to be distributed in the regions proximal to the centromeres of the chromosomes. The distribution of the Rim2/Hipa CACTA elements was surveyed in 15 different Oryza species via Rim2/Hipa-TD. While Rim2/Hipa-TD yielded ample amplification profiles between 100 to 700 bp in the AA diploid Oryza species, other species having BB, CC
Delprat, Alejandra; Negre, Bàrbara; Puig, Marta; Ruiz, Alfredo
Transposable elements (TEs) are responsible for the generation of chromosomal inversions in several groups of organisms. However, in Drosophila and other Dipterans, where inversions are abundant both as intraspecific polymorphisms and interspecific fixed differences, the evidence for a role of TEs is scarce. Previous work revealed that the transposon Galileo was involved in the generation of two polymorphic inversions of Drosophila buzzatii. To assess the impact of TEs in Drosophila chromosomal evolution and shed light on the mechanism involved, we isolated and sequenced the two breakpoints of another widespread polymorphic inversion from D. buzzatii, 2z(3). In the non inverted chromosome, the 2z(3) distal breakpoint was located between genes CG2046 and CG10326 whereas the proximal breakpoint lies between two novel genes that we have named Dlh and Mdp. In the inverted chromosome, the analysis of the breakpoint sequences revealed relatively large insertions (2,870-bp and 4,786-bp long) including two copies of the transposon Galileo (subfamily Newton), one at each breakpoint, plus several other TEs. The two Galileo copies: (i) are inserted in opposite orientation; (ii) present exchanged target site duplications; and (iii) are both chimeric. Our observations provide the best evidence gathered so far for the role of TEs in the generation of Drosophila inversions. In addition, they show unequivocally that ectopic recombination is the causative mechanism. The fact that the three polymorphic D. buzzatii inversions investigated so far were generated by the same transposon family is remarkable and is conceivably due to Galileo's unusual structure and current (or recent) transpositional activity.
Nouroz, F.; Noreen, S.; Harrison, H.
Among DNA transposons, PIF/Harbinger is most recently identified superfamily characterized by 3 bp target site duplications (TSDs), flanked by 14-45 bp terminal inverted repeats (TIRs) and displaying DDD or DDE domain displaying transposase. Their autonomous elements contain two open reading frames, ORF1 and ORF2 encoding superfamily specific transposase and DNA-binding domain. Harbinger DNA transposons are recently identified in few plants. In present study, computational and molecular approaches were used for the identification of 8 Harbinger transposons, of which only 2 were complete with putative trans posase, while rest 6 lack transposase and are considered as defective or non-autonomous elements. They ranged in size from 0.5-4 kb with 3 bp TSDs, 15-42 bp TIRs and internal AT richregions. The PCR amplification of Brassica Harbinger transposase revealed diversity and ancient nature of these elements. The amplification polymorphism of some non-autonomous Harbingers showed species specific distribution. Phylogenetic analyses of transposase clustered them into two clades (monocot and dicot) and five sub-clades. The Brassica, Arabidopsis and Malustransposase clustered into genera specific sub-clades; although a lot of homology in transposase was observed. The multiple sequence alignment of Brassica and related transposase showed homology in five conserved blocks. The DD/Sub 35/E triad and sequences showed similarity to already known Pong-like or Arabidopsis ATIS12 Harbinger transposase in contrast to other transposase having DD/Sub 47/E or DD/Sub 48/E motifs. The present study will be helpful in the characterization of Harbingers, their structural diversity in related genera and Harbinger based molecular markers for varietal/lines identifications. (author)
Chen, Li; Schmidt, Emmett V; Stuart, Lynda; Ohsumi, Toshiro K; Burgess, Shawn; Varshney, Gaurav K; Dastur, Anahita; Borowsky, Mark; Benes, Cyril; Lacy-Hulbert, Adam
The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. Here we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost. Using this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches. We have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens
Rezsohazy, R; van Luenen, H G; Durbin, R M; Plasterk, R H
We have found a novel transposon in the genome of Caenorhabditis elegans. Tc7 is a 921 bp element, made up of two 345 bp inverted repeats separated by a unique, internal sequence. Tc7 does not contain an open reading frame. The outer 38 bp of the inverted repeat show 36 matches with the outer 38 bp of Tc1. This region of Tc1 contains the Tc1-transposase binding site. Furthermore, Tc7 is flanked by TA dinucleotides, just like Tc1, which presumably correspond to the target duplication generated...
de Koning-Ward Tania F
Full Text Available Abstract The ability to analyze gene function in malaria-causing Plasmodium parasites has received a boost with a recent paper in BMC Genomics that describes a genome-wide mutagenesis system in the rodent malaria species Plasmodium berghei using the transposon piggyBac. This advance holds promise for identifying and validating new targets for intervention against malaria. But further improvements are still needed for the full power of genome-wide molecular genetic screens to be utilized in this organism. See research article: http://www.biomedcentral.com/1471-2164/12/155
Kieboom, J; Bruinenberg, R; Keizer-Gunnink, [No Value; de Bont, JAM
Fourteen solvent-sensitive transposon mutants were generated from the solvent-tolerant Pseudomonas putida strain S12 by applying the TnMOD-KmO mutagenesis system. These mutants were unable to grow in the presence of octanol and toluene. By cloning the region flanking the transposon insertion point a
Shah, Vandan; Pierre, Brennal; Kim, Jin Ryoun
Insertional fusion between multiple protein domains represents a novel means of creating integrated functionalities. Currently, there is no robust guideline for selection of insertion sites ensuring the desired functional outcome of insertional fusion. Therefore, construction and testing of random domain insertion libraries, in which a host protein domain is randomly inserted into a guest protein domain, significantly benefit extensive exploration of sequence spaces for insertion sites. Short peptide residues are usually introduced between protein domains to alleviate structural conflicts, and the interdomain linker residues may affect the functional outcome of protein insertion complexes. Unfortunately, optimal control of interdomain linker residues is not always available in conventional methods used to construct random domain insertion libraries. Moreover, most conventional methods employ blunt-end rather than sticky-end ligation between host and guest DNA fragments, thus lowering library construction efficiency. Here, we report the facile construction of random domain insertion libraries using an engineered transposon. We show that random domain insertion with optimal control of interdomain linker residues was possible with our engineered transposon-based method. In addition, our method employs sticky-end rather than blunt-end ligation between host and guest DNA fragments, thus allowing for facile construction of relatively large sized libraries. Copyright © 2012 Elsevier Inc. All rights reserved.
Vilain, Sven; Vanhauwaert, Roeland; Maes, Ine; Schoovaerts, Nils; Zhou, Lujia; Soukup, Sandra; da Cunha, Raquel; Lauwers, Elsa; Fiers, Mark; Verstreken, Patrik
Modern molecular genetics studies necessitate the manipulation of genes in their endogenous locus, but most of the current methodologies require an inefficient donor-dependent homologous recombination step to locally modify the genome. Here we describe a methodology to efficiently generate Drosophila knock-in alleles by capitalizing on the availability of numerous genomic MiMIC transposon insertions carrying recombinogenic attP sites. Our methodology entails the efficient PhiC31-mediated integration of a recombination cassette flanked by unique I-SceI and/or I-CreI restriction enzyme sites into an attP-site. These restriction enzyme sites allow for double-strand break-mediated removal of unwanted flanking transposon sequences, while leaving the desired genomic modifications or recombination cassettes. As a proof-of-principle, we mutated LRRK, tau, and sky by using different MiMIC elements. We replaced 6 kb of genomic DNA encompassing the tau locus and 35 kb encompassing the sky locus with a recombination cassette that permits easy integration of DNA at these loci and we also generated a functional LRRK(HA) knock in allele. Given that ~92% of the Drosophila genes are located within the vicinity (MiMIC element, our methodology enables the efficient manipulation of nearly every locus in the fruit fly genome without the need for inefficient donor-dependent homologous recombination events. Copyright © 2014 Vilain et al.
Venken, Koen J. T.; Schulze, Karen L.; Haelterman, Nele A.; Pan, Hongling; He, Yuchun; Evans-Holm, Martha; Carlson, Joseph W.; Levis, Robert W.; Spradling, Allan C.; Hoskins, Roger A.; Bellen, Hugo J.
We demonstrate the versatility of a collection of insertions of the transposon Minos mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 attP sites. MiMIC integrates almost at random in the genome to create sites for DNA manipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp system. Insertions within coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the Drosophila melanogaster toolkit. PMID:21985007
Bellen, Hugo J.; Levis, Robert W.; Liao, Guochun; He, Yuchun; Carlson, Joseph W.; Tsang, Garson; Evans-Holm, Martha; Hiesinger, P. Robin; Schulze, Karen L.; Rubin, Gerald M.; Hoskins, Roger A.; Spradling, Allan C.
The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in more than 30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6,300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7,140 lines. It now includes individual lines predicted to disrupt 5,362 of the 13,666 currently annotated Drosophila genes (39 percent). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene mis-expression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.
Kim, Hyun-Soo [Ewha Womans Univ., Seoul (Korea, Republic of). Dept. of Life Sciences; Kim, Na-Rae [Ewha Womans Univ., Seoul (Korea, Republic of). Div. of Life and Pharmaceutical Sciences; Yang, Jungwoo [Ewha Womans Univ., Seoul (Korea, Republic of). Microbial Resources Research Center; Choi, Wonja [Ewha Womans Univ., Seoul (Korea, Republic of). Dept. of Life Sciences; Ewha Womans Univ., Seoul (Korea, Republic of). Div. of Life and Pharmaceutical Sciences; Ewha Womans Univ., Seoul (Korea, Republic of). Microbial Resources Research Center
Saccharomyces cerevisiae strains tolerant to ethanol and heat stresses are important for industrial ethanol production. In this study, five strains (Tn 1-5) tolerant to up to 15% ethanol were isolated by screening a transposon-mediated mutant library. Two of them displayed tolerance to heat (42 C). The determination of transposon insertion sites and Northern blot analysis identified seven putative genes (CMP2, IMD4, SSK2, PPG1, DLD3, PAM1, and MSN2) and revealed simultaneous down-regulations of CMP2 and IMD4, and SSK2 and PPG1, down-regulation of DLD3, and disruptions of the open reading frame of PAM1 and MSN2, indicating that ethanol and/or heat tolerance can be conferred. Knockout mutants of these seven individual genes were ethanol tolerant and three of them (SSK2, PPG1, and PAM1) were tolerant to heat. Such tolerant phenotypes reverted to sensitive phenotypes by the autologous or overexpression of each gene. Five transposon mutants showed higher ethanol production and grew faster than the control strain when cultured in rich media containing 30% glucose and initial 6% ethanol at 30 C. Of those, two thermotolerant transposon mutants (Tn 2 and Tn 3) exhibited significantly enhanced growth and ethanol production compared to the control at 42 C. The genes identified in this study may provide a basis for the application in developing industrial yeast strains. (orig.)
Jacobs, Gunnar; Dechyeva, Daryna; Menzel, Gerhard; Dombrowski, Cora; Schmidt, Thomas
Transposons of the Tc1-mariner superfamily are widespread in eukaryotic genomes. We have isolated the mariner element Vulmar1 from Beta vulgaris L., which is 3909 bp long and bordered by perfect terminal inverted repeats of 32 bp with homology to terminal inverted repeats of transposons from soybean and rice. According to a characteristic amino acid signature, Vulmar1 can be assigned to the DD39D group of mariner transposons. Vulmar1 is flanked by a 5'-TA-3' target site duplication that is typical for mariner transposons. Southern hybridization revealed that mariner-like copies are highly abundant in Beta species, and sequence analysis of 10 transposase fragments from representative species of the four Beta sections revealed an identity between 34% and 100% after conceptual translation. By fluorescent in situ hybridization, Vulmar1 was detected in distal euchromatin as well as in some intercalary and pericentromeric regions of all B. vulgaris chromosomes. In addition, using PCR, we were able to amplify fragments of the transposase gene of En/Spm-like transposons in the genus Beta. En/Spm-like transposase sequences are highly amplified in four Beta sections and showed a considerable degree of conservation (88.5-100%) at the protein level, while the homology to corresponding regions of En/Spm transposons of other plant species ranges from 49.5% to 62.5%. By fluorescent in situ hybridization, En/Spm-like transposon signals of strong intensity were detected on all chromosomes of B. vulgaris.
Lorenzen, Marcé D; Gnirke, Andreas; Margolis, Jonathan; Garnes, Jeffrey; Campbell, Margie; Stuart, Jeffrey J; Aggarwal, Rajat; Richards, Stephen; Park, Yoonseong; Beeman, Richard W
Maternal-Effect Dominant Embryonic Arrest ("Medea") factors are selfish nuclear elements that combine maternal-lethal and zygotic-rescue activities to gain a postzygotic survival advantage. We show that Medea(1) activity in Tribolium castaneum is associated with a composite Tc1 transposon inserted just downstream of the neurotransmitter reuptake symporter bloated tubules (blot), whose Drosophila ortholog has both maternal and zygotic functions. The 21.5-kb insertion contains defective copies of elongation initiation factor-3, ATP synthase subunit C, and an RNaseD-related gene, as well as a potentially intact copy of a prokaryotic DUF1703 gene. Sequence comparisons suggest that the current distribution of Medea(1) reflects global emanation after a single transpositional event in recent evolutionary time. The Medea system in Tribolium represents an unusual type of intragenomic conflict and could provide a useful vehicle for driving desirable genes into populations.
Gonçalves, Juliana W; Valiati, Victor Hugo; Delprat, Alejandra; Valente, Vera L S; Ruiz, Alfredo
Galileo is one of three members of the P superfamily of DNA transposons. It was originally discovered in Drosophila buzzatii, in which three segregating chromosomal inversions were shown to have been generated by ectopic recombination between Galileo copies. Subsequently, Galileo was identified in six of 12 sequenced Drosophila genomes, indicating its widespread distribution within this genus. Galileo is strikingly abundant in Drosophila willistoni, a neotropical species that is highly polymorphic for chromosomal inversions, suggesting a role for this transposon in the evolution of its genome. We carried out a detailed characterization of all Galileo copies present in the D. willistoni genome. A total of 191 copies, including 133 with two terminal inverted repeats (TIRs), were classified according to structure in six groups. The TIRs exhibited remarkable variation in their length and structure compared to the most complete copy. Three copies showed extended TIRs due to internal tandem repeats, the insertion of other transposable elements (TEs), or the incorporation of non-TIR sequences into the TIRs. Phylogenetic analyses of the transposase (TPase)-encoding and TIR segments yielded two divergent clades, which we termed Galileo subfamilies V and W. Target-site duplications (TSDs) in D. willistoni Galileo copies were 7- or 8-bp in length, with the consensus sequence GTATTAC. Analysis of the region around the TSDs revealed a target site motif (TSM) with a 15-bp palindrome that may give rise to a stem-loop secondary structure. There is a remarkable abundance and diversity of Galileo copies in the D. willistoni genome, although no functional copies were found. The TIRs in particular have a dynamic structure and extend in different ways, but their ends (required for transposition) are more conserved than the rest of the element. The D. willistoni genome harbors two Galileo subfamilies (V and W) that diverged ~9 million years ago and may have descended from an ancestral
Full Text Available Transposable elements (TEs are the most abundant group of genomic elements in plants that can be found in genic or intergenic regions of their host genomes. Several stimuli such as biotic or abiotic stress have roles in either activating their transcription or transposition. Here the effect of the Panicum mosaic virus (PMV and its satellite virus (SPMV infection on the transposon transcription of the Brachypodium distachyon model plant was investigated. To evaluate the transcription activity of TEs, transcriptomic data of mock and virus inoculated plants were compared. Our results indicate that major components of TEs are retroelements in all RNA-seq libraries. The number of transcribed TEs detected in mock inoculated plants is higher than virus inoculated plants. In comparison with mock inoculated plants 13% of the TEs showed at least two folds alteration upon PMV infection and 21% upon PMV+SPMV infection. Rather than inoculation with PMV alone inoculation with PMV+SPMV together also increased various TE encoding transcripts expressions. MuDR-N78C_OS encoding transcript was strongly up-regulated against both PMV and PMV+SPMV infection. The synergism generated by PMV and SPMV together enhanced TE transcripts expressions than PMV alone. It was observed that viral infection induced the transcriptional activity of several transposons. The results suggest that increased expressions of TEs might have a role in response to biotic stress in B. distachyon. Identification of TEs which are taking part in stress can serve useful information for functional genomics and designing novel breeding strategies in developing stress resistance crops.
Loftus, Brendan J.; Fung, Eula; Roncaglia, Paola; Rowley, Don; Amedeo, Paolo; Bruno, Dan; Vamathevan, Jessica; Miranda, Molly; Anderson, Iain J.; Fraser, James A.; Allen, Jonathan E.; Bosdet, Ian E.; Brent, Michael R.; Chiu, Readman; Doering, Tamara L.
Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its ~20-megabase genome, which contains ~6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype i...
Murata, M; Uchida, T; Yang, Y; Lezhava, A; Kinashi, H
We have comprehensively analyzed the linear chromosomes of Streptomyces griseus mutants constructed and kept in our laboratory. During this study, macrorestriction analysis of AseI and DraI fragments of mutant 402-2 suggested a large chromosomal inversion. The junctions of chromosomal inversion were cloned and sequenced and compared with the corresponding target sequences in the parent strain 2247. Consequently, a transposon-involved mechanism was revealed. Namely, a transposon originally located at the left target site was replicatively transposed to the right target site in an inverted direction, which generated a second copy and at the same time caused a 2.5-Mb chromosomal inversion. The involved transposon named TnSGR was grouped into a new subfamily of the resolvase-encoding Tn3 family transposons based on its gene organization. At the end, terminal diversity of S. griseus chromosomes is discussed by comparing the sequences of strains 2247 and IFO13350.
Hodge, Russ; Narayanavari, Suneel A; Izsvák, Zsuzsanna; Ivics, Zoltán
Gene therapies will only become a widespread tool in the clinical treatment of human diseases with the advent of gene transfer vectors that integrate genetic information stably, safely, effectively, and economically. Two decades after the discovery of the Sleeping Beauty (SB) transposon, it has been transformed into a vector system that is fulfilling these requirements. SB may well overcome some of the limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are being used in the majority of ongoing clinical trials. The SB system has achieved a high level of stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, representing crucial steps that may permit its clinical use in the near future. This article reviews the most important aspects of SB as a tool for gene therapy, including aspects of its vectorization and genomic integration. As an illustration, the clinical development of the SB system toward gene therapy of age-related macular degeneration and cancer immunotherapy is highlighted.
Yang, Guanhua; Billings, Gabriel; Hubbard, Troy P; Park, Joseph S; Yin Leung, Ka; Liu, Qin; Davis, Brigid M; Zhang, Yuanxing; Wang, Qiyao; Waldor, Matthew K
Transposon insertion sequencing (TIS) is a powerful high-throughput genetic technique that is transforming functional genomics in prokaryotes, because it enables genome-wide mapping of the determinants of fitness. However, current approaches for analyzing TIS data assume that selective pressures are constant over time and thus do not yield information regarding changes in the genetic requirements for growth in dynamic environments (e.g., during infection). Here, we describe structured analysis of TIS data collected as a time series, termed pattern analysis of conditional essentiality (PACE). From a temporal series of TIS data, PACE derives a quantitative assessment of each mutant's fitness over the course of an experiment and identifies mutants with related fitness profiles. In so doing, PACE circumvents major limitations of existing methodologies, specifically the need for artificial effect size thresholds and enumeration of bacterial population expansion. We used PACE to analyze TIS samples of Edwardsiella piscicida (a fish pathogen) collected over a 2-week infection period from a natural host (the flatfish turbot). PACE uncovered more genes that affect E. piscicida 's fitness in vivo than were detected using a cutoff at a terminal sampling point, and it identified subpopulations of mutants with distinct fitness profiles, one of which informed the design of new live vaccine candidates. Overall, PACE enables efficient mining of time series TIS data and enhances the power and sensitivity of TIS-based analyses. IMPORTANCE Transposon insertion sequencing (TIS) enables genome-wide mapping of the genetic determinants of fitness, typically based on observations at a single sampling point. Here, we move beyond analysis of endpoint TIS data to create a framework for analysis of time series TIS data, termed pattern analysis of conditional essentiality (PACE). We applied PACE to identify genes that contribute to colonization of a natural host by the fish pathogen
Full Text Available Mitochondria execute key pathways of central metabolism and serve as cellular sensing and signaling entities, functions that depend upon interactions between mitochondrial and nuclear genetic systems. This is exemplified in cytoplasmic male sterility type S (CMS-S of Zea mays, where novel mitochondrial open reading frames are associated with a pollen collapse phenotype, but nuclear restorer-of-fertility (restorer mutations rescue pollen function. To better understand these genetic interactions, we screened Activator-Dissociation (Ac-Ds, Enhancer/Suppressor-mutator (En/Spm, and Mutator (Mu transposon-active CMS-S stocks to recover new restorer mutants. The frequency of restorer mutations increased in transposon-active stocks compared to transposon-inactive stocks, but most mutants recovered from Ac-Ds and En/Spm stocks were unstable, reverting upon backcrossing to CMS-S inbred lines. However, 10 independent restorer mutations recovered from CMS-S Mu transposon stocks were stable upon backcrossing. Many restorer mutations condition seed-lethal phenotypes that provide a convenient test for allelism. Eight such mutants recovered in this study included one pair of allelic mutations that were also allelic to the previously described rfl2-1 mutant. Targeted analysis of mitochondrial proteins by immunoblot identified two features that consistently distinguished restored CMS-S pollen from comparably staged, normal-cytoplasm, nonmutant pollen: increased abundance of nuclear-encoded alternative oxidase relative to mitochondria-encoded cytochrome oxidase and decreased abundance of mitochondria-encoded ATP synthase subunit 1 compared to nuclear-encoded ATP synthase subunit 2. CMS-S restorer mutants thus revealed a metabolic plasticity in maize pollen, and further study of these mutants will provide new insights into mitochondrial functions that are critical to pollen and seed development.
Fjord-Larsen, L; Kusk, Poul Henrik; Emerich, D F
transposon expression technology to establish a new clinical grade cell line, NGC0211, with at least 10 times higher NGF production than that of NGC-0295. To test whether encapsulation of this cell line provides a relevant dose escalation step in delivering NGF for treatment of the cognitive decline in AD...... cases correlate with highly improved potency.Gene Therapy advance online publication, 24 November 2011; doi:10.1038/gt.2011.178....
Liu, Hongyan; Wang, Guangce
Based on the transposon-mutagenized library of Pantoea agglomerans BH18, mutant screens were conducted to obtain the strain with the highest Fe (III) reduction and hydrogen production. Of these transposon-mutagenized mutants, the mutant strain TB230 was screened for high Fe (III)-reducing efficiency and hydrogen production. The PCR amplification and kanamycin resistance selection results indicated that the transposon insertion of the mutant strain TB230 was stable. Hydrogen production of the mutant strain TB230 was (2.21 ± 0.34) mol H 2 /mol glucose, which increased hydrogen production by over 40% compared with that of the wild type strain. The accumulation concentration of Fe (II) in the medium of the mutant strain TB230 with Fe (OH) 3 as the sole electron acceptor was (7.39 ± 0.49) mmol/l, which was approximately 3-fold greater than that of the wild type strain. The mutant strain TB230 showed high Fe (III)-reducing activity and hydrogen production by adopting glucose and pyruvate as the carbon source. In addition, the mutant strain TB230 was capable of Fe (III) reduction and hydrogen production under fresh or marine conditions. This result indicates that the mutant strain with high microbial Fe (III) reduction and hydrogen production is beneficial for the improvement of anaerobic performance. - Highlights: • The mutant strain TB230 was a transposon-mutagenized strain of Pantoea agglomerans BH18. • Strain TB230 was screened for high Fe (III)-reducing efficiency and hydrogen production. • H 2 yield and Fe (III)-reducing activity were 2.21 ± 0.34 and 7.39 ± 0.49 in marine condition. • Strain TB230 was capable of Fe (III) reduction and hydrogen production in fresh or marine condition
Li, Yubin; Harris, Linda; Dooner, Hugo K.
Mutator (Mu) elements, one of the most diverse superfamilies of DNA transposons, are found in all eukaryotic kingdoms, but are particularly numerous in plants. Most of the present knowledge on the transposition behavior of this superfamily comes from studies of the maize (Zea mays) Mu elements, whose transposition is mediated by the autonomous Mutator-Don Robertson (MuDR) element. Here, we describe the maize element TED (for Transposon Ellen Dempsey), an autonomous cousin that differs significantly from MuDR. Element excision and reinsertion appear to require both proteins encoded by MuDR, but only the single protein encoded by TED. Germinal excisions, rare with MuDR, are common with TED, but arise in one of the mitotic divisions of the gametophyte, rather than at meiosis. Instead, transposition-deficient elements arise at meiosis, suggesting that the double-strand breaks produced by element excision are repaired differently in mitosis and meiosis. Unlike MuDR, TED is a very low-copy transposon whose number and activity do not undergo dramatic changes upon inbreeding or outcrossing. Like MuDR, TED transposes mostly to unlinked sites and can form circular transposition products. Sequences closer to TED than to MuDR were detected only in the grasses, suggesting a rather recent evolutionary split from a common ancestor. PMID:24038653
Li, Yubin; Harris, Linda; Dooner, Hugo K
Mutator (Mu) elements, one of the most diverse superfamilies of DNA transposons, are found in all eukaryotic kingdoms, but are particularly numerous in plants. Most of the present knowledge on the transposition behavior of this superfamily comes from studies of the maize (Zea mays) Mu elements, whose transposition is mediated by the autonomous Mutator-Don Robertson (MuDR) element. Here, we describe the maize element TED (for Transposon Ellen Dempsey), an autonomous cousin that differs significantly from MuDR. Element excision and reinsertion appear to require both proteins encoded by MuDR, but only the single protein encoded by TED. Germinal excisions, rare with MuDR, are common with TED, but arise in one of the mitotic divisions of the gametophyte, rather than at meiosis. Instead, transposition-deficient elements arise at meiosis, suggesting that the double-strand breaks produced by element excision are repaired differently in mitosis and meiosis. Unlike MuDR, TED is a very low-copy transposon whose number and activity do not undergo dramatic changes upon inbreeding or outcrossing. Like MuDR, TED transposes mostly to unlinked sites and can form circular transposition products. Sequences closer to TED than to MuDR were detected only in the grasses, suggesting a rather recent evolutionary split from a common ancestor.
Full Text Available The term water stress refers to the effects of low water availability on microbial growth and physiology. Water availability has been proposed as a major constraint for the use of microorganisms in contaminated sites with the purpose of bioremediation. Sphingomonas wittichii RW1 is a bacterium capable of degrading the xenobiotic compounds dibenzofuran and dibenzo-p-dioxin, and has potential to be used for targeted bioremediation. The aim of the current work was to identify genes implicated in water stress in RW1 by means of transposon mutagenesis and mutant growth experiments. Conditions of low water potential were mimicked by adding NaCl to the growth media. Three different mutant selection or separation method were tested, which, however recovered different mutants. Recovered transposon mutants with poorer growth under salt-induced water stress carried insertions in genes involved in proline and glutamate biosynthesis, and further in a gene putatively involved in aromatic compound catabolism. Transposon mutants growing poorer on medium with lowered water potential also included ones that had insertions in genes involved in more general functions such as transcriptional regulation, elongation factor, cell division protein, RNA polymerase β or an aconitase.
Marcus, Jeffrey M; Hughes, Tia M
Structured inquiry approaches, in which students receive a Drosophila strain of unknown genotype to analyze and map the constituent mutations, are a common feature of many genetics teaching laboratories. The required crosses frustrate many students because they are aware that they are participating in a fundamentally trivial exercise, as the map locations of the genes are already established and have been recalculated thousands of times by generations of students. We modified the traditional structured inquiry approach to include a novel research experience for the students in our undergraduate genetics laboratories. Students conducted crosses with Drosophila strains carrying P[lacW] transposon insertions in genes without documented recombination map positions, representing a large number of unique, but equivalent genetic unknowns. Using the eye color phenotypes associated with the inserts as visible markers, it is straightforward to calculate recombination map positions for the interrupted loci. Collectively, our students mapped 95 genetic loci on chromosomes 2 and 3. In most cases, the calculated 95% confidence interval for meiotic map location overlapped with the predicted map position based on cytology. The research experience evoked positive student responses and helped students better understand the nature of scientific research for little additional cost or instructor effort.
Lin, Yu-Mei; Chou, I-Chun; Wang, Jaw-Fen; Ho, Fang-I; Chu, Yu-Ju; Huang, Pei-Cheng; Lu, Der-Kang; Shen, Hwei-Ling; Elbaz, Mounira; Huang, Shu-Mei; Cheng, Chiu-Ping
Ralstonia solanacearum causes a deadly wilting disease on a wide range of crops. To elucidate pathogenesis of this bacterium in different host plants, we set out to identify R. solanacearum genes involved in pathogenesis by screening random transposon insertion mutants of a highly virulent strain, Pss190, on tomato and Arabidopsis thaliana. Mutants exhibiting various decreased virulence levels on these two hosts were identified. Sequence analysis showed that most, but not all, of the identified pathogenesis genes are conserved among distinct R. solanacearum strains. A few of the disrupted loci were not reported previously as being involved in R. solanacearum pathogenesis. Notably, a group of mutants exhibited differential pathogenesis on tomato and Arabidopsis. These results were confirmed by characterizing allelic mutants in one other R. solanacearum strain of the same phylotype. The significantly decreased mutants' colonization in Arabidopsis was found to be correlated with differential pathogenesis on these two plants. Differential requirement of virulence genes suggests adaptation of this bacterium in different host environments. Together, this study reveals commonalities and differences of R. solanacearum pathogenesis on single solanaceous and nonsolanaceous hosts, and provides important new insights into interactions between R. solanacearum and different host plants.
Full Text Available Transposon insertion sequencing (TIS is a powerful high-throughput genetic technique that is transforming functional genomics in prokaryotes, because it enables genome-wide mapping of the determinants of fitness. However, current approaches for analyzing TIS data assume that selective pressures are constant over time and thus do not yield information regarding changes in the genetic requirements for growth in dynamic environments (e.g., during infection. Here, we describe structured analysis of TIS data collected as a time series, termed pattern analysis of conditional essentiality (PACE. From a temporal series of TIS data, PACE derives a quantitative assessment of each mutant’s fitness over the course of an experiment and identifies mutants with related fitness profiles. In so doing, PACE circumvents major limitations of existing methodologies, specifically the need for artificial effect size thresholds and enumeration of bacterial population expansion. We used PACE to analyze TIS samples of Edwardsiella piscicida (a fish pathogen collected over a 2-week infection period from a natural host (the flatfish turbot. PACE uncovered more genes that affect E. piscicida’s fitness in vivo than were detected using a cutoff at a terminal sampling point, and it identified subpopulations of mutants with distinct fitness profiles, one of which informed the design of new live vaccine candidates. Overall, PACE enables efficient mining of time series TIS data and enhances the power and sensitivity of TIS-based analyses.
Younger, Andrew K D; Su, Peter Y; Shepard, Andrea J; Udani, Shreya V; Cybulski, Thaddeus R; Tyo, Keith E J; Leonard, Joshua N
Naturally evolved metabolite-responsive biosensors enable applications in metabolic engineering, ranging from screening large genetic libraries to dynamically regulating biosynthetic pathways. However, there are many metabolites for which a natural biosensor does not exist. To address this need, we developed a general method for converting metabolite-binding proteins into metabolite-responsive transcription factors-Biosensor Engineering by Random Domain Insertion (BERDI). This approach takes advantage of an in vitro transposon insertion reaction to generate all possible insertions of a DNA-binding domain into a metabolite-binding protein, followed by fluorescence activated cell sorting to isolate functional biosensors. To develop and evaluate the BERDI method, we generated a library of candidate biosensors in which a zinc finger DNA-binding domain was inserted into maltose binding protein, which served as a model well-studied metabolite-binding protein. Library diversity was characterized by several methods, a selection scheme was deployed, and ultimately several distinct and functional maltose-responsive transcriptional biosensors were identified. We hypothesize that the BERDI method comprises a generalizable strategy that may ultimately be applied to convert a wide range of metabolite-binding proteins into novel biosensors for applications in metabolic engineering and synthetic biology. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: firstname.lastname@example.org.
Di Giovanni, Michela; Cenci, Alberto; Janni, Michela; D'Ovidio, Renato
Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defence. Wheat pgip genes have been isolated from the B (Tapgip1) and D (Tapgip2) genomes, and now we report the identification of pgip genes from the A genomes of wild and cultivated wheats. By Southern blots and sequence analysis of BAC clones we demonstrated that wheat contains a single copy pgip gene per genome and the one from the A genome, pgip3, is inactivated by the insertion of a long terminal repeat copia retrotranspon within the fourth LRR. We demonstrated also that this retrotransposon insertion is present in Triticum urartu and all the polyploidy wheats assayed, but is absent in T. monococcum (Tmpgip3), suggesting that this insertion took place after the divergence between T. monococcum and T. urartu, but before the formation of the polyploid wheats. We identified also two independent insertion events of new Class II transposable elements, Vacuna, belonging to the Mutator superfamily, that interrupted the Tdipgip1 gene of T. turgidum ssp. dicoccoides. The occurrence of these transposons within the coding region of Tdipgip1 facilitated the mapping of the Pgip locus in the pericentric region of the short arm of chromosome group 7. We speculate that the inactivation of pgip genes are tolerated because of redundancy of PGIP activities in the wheat genome.
Full Text Available BACKGROUND: CTX-M-producing Escherichia coli strains are regarded as major global pathogens. METHODOLOGY/PRINCIPAL FINDINGS: The nucleotide sequence of three plasmids (pEC_B24: 73801-bp; pEC_L8: 118525-bp and pEC_L46: 144871-bp from Escherichia coli isolates obtained from patients with urinary tract infections and one plasmid (pEC_Bactec: 92970-bp from an Escherichia coli strain isolated from the joint of a horse with arthritis were determined. Plasmid pEC_Bactec belongs to the IncI1 group and carries two resistance genes: bla(TEM-1 and bla(CTX-M-15. It shares more than 90% homology with a previously published bla(CTX-M-plasmid from E. coli of human origin. Plasmid pEC_B24 belongs to the IncFII group whereas plasmids pEC_L8 and pEC_L46 represent a fusion of two replicons of type FII and FIA. On the pEC_B24 backbone, two resistance genes, bla(TEM-1 and bla(CTX-M-15, were found. Six resistance genes, bla(TEM-1, bla(CTX-M-15, bla(OXA-1, aac6'-lb-cr, tetA and catB4, were detected on the pEC_L8 backbone. The same antimicrobial drug resistance genes, with the exception of tetA, were also identified on the pEC_L46 backbone. Genome analysis of all 4 plasmids studied provides evidence of a seemingly frequent transposition event of the bla(CTX-M-15-ISEcp1 element. This element seems to have a preferred insertion site at the tnpA gene of a bla(TEM-carrying Tn3-like transposon, the latter itself being inserted by a transposition event. The IS26-composite transposon, which contains the bla(OXA-1, aac6'-lb-cr and catB4 genes, was inserted into plasmids pEC_L8 and pEC_L46 by homologous recombination rather than a transposition event. Results obtained for pEC_L46 indicated that IS26 also plays an important role in structural rearrangements of the plasmid backbone and seems to facilitate the mobilisation of fragments from other plasmids. CONCLUSIONS: Collectively, these data suggests that IS26 together with ISEcp1 could play a critical role in the evolution of
Full Text Available The recognition of a positive correlation between organism genome size with its transposable element (TE content, represents a key discovery of the field of genome biology. Considerable evidence accumulated since then suggests the involvement of TEs in genome structure, evolution and function. The global genome reorganization brought about by transposon activity might play an adaptive/regulatory role in the host response to environmental challenges, reminiscent of McClintock’s original ’Controlling Element’ hypothesis. This regulatory aspect of TEs is also garnering support in light of the recent evidences which project TEs as distributed genomic control modules. According to this view, TEs are capable of actively reprogramming host genes circuits and ultimately fine-tuning the host response to specific environmental stimuli. Moreover, the stress-induced changes in epigenetic status of TE activity may allow TEs to propagate their stress responsive elements to host genes; the resulting genome fluidity can permit phenotypic plasticity and adaptation to stress. Given their predominating presence in the plant genomes, nested organization in the genic regions and potential regulatory role in stress response, TEs hold unexplored potential for crop improvement programs. This review intends to present the current information about the roles played by TEs in plant genome organization, evolution and function, and highlight the regulatory mechanisms in plant stress responses. We will also briefly discuss the connection between TE activity, host epigenetic response and phenotypic plasticity as a critical link for traversing the translational bridge from a purely basic study of TEs, to the applied field of stress adaptation and crop improvement.
Negi, Pooja; Rai, Archana N; Suprasanna, Penna
The recognition of a positive correlation between organism genome size with its transposable element (TE) content, represents a key discovery of the field of genome biology. Considerable evidence accumulated since then suggests the involvement of TEs in genome structure, evolution and function. The global genome reorganization brought about by transposon activity might play an adaptive/regulatory role in the host response to environmental challenges, reminiscent of McClintock's original 'Controlling Element' hypothesis. This regulatory aspect of TEs is also garnering support in light of the recent evidences, which project TEs as "distributed genomic control modules." According to this view, TEs are capable of actively reprogramming host genes circuits and ultimately fine-tuning the host response to specific environmental stimuli. Moreover, the stress-induced changes in epigenetic status of TE activity may allow TEs to propagate their stress responsive elements to host genes; the resulting genome fluidity can permit phenotypic plasticity and adaptation to stress. Given their predominating presence in the plant genomes, nested organization in the genic regions and potential regulatory role in stress response, TEs hold unexplored potential for crop improvement programs. This review intends to present the current information about the roles played by TEs in plant genome organization, evolution, and function and highlight the regulatory mechanisms in plant stress responses. We will also briefly discuss the connection between TE activity, host epigenetic response and phenotypic plasticity as a critical link for traversing the translational bridge from a purely basic study of TEs, to the applied field of stress adaptation and crop improvement.
Full Text Available This study assessed the abundance of microsatellites, or simple sequence repeats (SSR, in 19 Eucalyptus EST libraries from FORESTs, containing cDNA sequences from five species: E. grandis, E. globulus, E. saligna, E. urophylla and E. camaldulensis. Overall, a total of 11,534 SSRs and 8,447 SSR-containing sequences (25.5% of total ESTs were identified, with an average of 1 SSR/2.5 kb when considering all motifs and 1 SSR/3.1 kb when mononucleotides were not included. Dimeric repeats were the most abundant (41.03%, followed by trimerics (36.11% and monomerics (19.59%. The most frequent motifs were A/T (87.24% for monomerics, AG/CT (94.44% for dimerics, CCG/CGG (37.87% for trimerics, AAGG/CCTT (18.75% for tetramerics, AGAGG/CCTCT (14.04% for pentamerics and ACGGCG/CGCCGT (6.30% for hexamerics. According to sequence length, Class II or potentially variable markers were the most commonly found, followed by Class III. Two sequences presented high similarity to previously published Eucalyptus sequences from the NCBI database, EMBRA_72 and EMBRA_122. Local blastn search for transposons did not reveal the presence of any transposable elements with a cut-off value of 10-50. The large number of microsatellites identified will contribute to the refinement of marker-assisted mapping and to the discovery of novel markers for virtually all genes of economic interest.
Full Text Available Pathogenic bacterial persisters are responsible for the recalcitrance of chronic and persistent infections to antimicrobial therapy. Although the mechanisms of persister formation and survival have been widely studied in Escherichia coli, persistence mechanisms in S. aureus remain largely unknown. Here, we screened a transposon mutant library of a clinical methicillin-resistant Staphylococcus aureus（MRSA）strain, USA500 (ST8, under antibiotic pressure and identified 13 genes whose insertion mutations resulted in a defect in persistence. These candidate genes were further confirmed by evaluating the survival of the mutants upon exposure to levofloxacin and several other stress conditions. We found 13 insertion mutants with significantly lower persister numbers under several stress conditions, including sdhA, sdhB, ureG, mnhG1, fbaA, ctaB, clpX, parE, HOU_0223, HOU_0587, HOU_2091, HOU_2315 and HOU_2346, which mapped into pathways of oxidative phosphorylation, TCA cycle, glycolysis, cell cycle and ABC transporters, suggesting that these genes and pathways may play an important role in persister formation and survival. The newly constructed knockout strains of ureG, sdhA and sdhB and their complemented strains were also tested for defect in persisters following exposure to levofloxacin and several other stress conditions. The results from these experiments were consistent with the screening results, which indicated that deletion of these genes in MRSA USA500 leads to persister defect. These findings provide novel insights into the mechanisms of persister formation and survival in S. aureus and offer new targets for the development of persister-directed antibiotics for the improved treatment of chronic and persistent infections.
Cavallini, Andrea; Natali, Lucia; Zuccolo, Andrea; Giordani, Tommaso; Jurman, Irena; Ferrillo, Veronica; Vitacolonna, Nicola; Sarri, Vania; Cattonaro, Federica; Ceccarelli, Marilena; Cionini, Pier Giorgio; Morgante, Michele
A sample-sequencing strategy combined with slot-blot hybridization and FISH was used to study the composition of the repetitive component of the sunflower genome. One thousand six hundred thirty-eight sequences for a total of 954,517 bp were analyzed. The fraction of sequences that can be classified as repetitive using computational and hybridization approaches amounts to 62% in total. Almost two thirds remain as yet uncharacterized in nature. Of those characterized, most belong to the gypsy superfamily of LTR-retrotransposons. Unlike in other species, where single families can account for large fractions of the genome, it appears that no transposon family has been amplified to very high levels in sunflower. All other known classes of transposable elements were also found. One family of unknown nature (contig 61) was the most repeated in the sunflower genome. The evolution of the repetitive component in the Helianthus genus and in other Asteraceae was studied by comparative analysis of the hybridization of total genomic DNAs from these species to the sunflower small-insert library and compared to gene-based phylogeny. Very little similarity is observed between Helianthus species and two related Asteraceae species outside of the genus. Most repetitive elements are similar in annual and perennial Helianthus species indicating that sequence amplification largely predates such divergence. Gypsy-like elements are more represented in the annuals than in the perennials, while copia-like elements are similarly represented, attesting a different amplification history of the two superfamilies of LTR-retrotransposons in the Helianthus genus.
Negi, Pooja; Rai, Archana N.; Suprasanna, Penna
The recognition of a positive correlation between organism genome size with its transposable element (TE) content, represents a key discovery of the field of genome biology. Considerable evidence accumulated since then suggests the involvement of TEs in genome structure, evolution and function. The global genome reorganization brought about by transposon activity might play an adaptive/regulatory role in the host response to environmental challenges, reminiscent of McClintock's original ‘Controlling Element’ hypothesis. This regulatory aspect of TEs is also garnering support in light of the recent evidences, which project TEs as “distributed genomic control modules.” According to this view, TEs are capable of actively reprogramming host genes circuits and ultimately fine-tuning the host response to specific environmental stimuli. Moreover, the stress-induced changes in epigenetic status of TE activity may allow TEs to propagate their stress responsive elements to host genes; the resulting genome fluidity can permit phenotypic plasticity and adaptation to stress. Given their predominating presence in the plant genomes, nested organization in the genic regions and potential regulatory role in stress response, TEs hold unexplored potential for crop improvement programs. This review intends to present the current information about the roles played by TEs in plant genome organization, evolution, and function and highlight the regulatory mechanisms in plant stress responses. We will also briefly discuss the connection between TE activity, host epigenetic response and phenotypic plasticity as a critical link for traversing the translational bridge from a purely basic study of TEs, to the applied field of stress adaptation and crop improvement. PMID:27777577
Bogumil Jacek Karas
Full Text Available With only hundreds of genes contained within their genomes, mycoplasmas have become model organisms for precise understanding of cellular processes, as well as platform organisms for predictable engineering of microbial functions for mission-critical applications. Despite the availability of whole genome writing in Mycoplasma mycoides, some traditional methods for genetic engineering are underdeveloped in mycoplasmas. Here we demonstrate two facile transposon-mediated approaches for introducing genes into the synthetic cell based on M. mycoides. The marker-less approach involves preparing a fragment containing only a small genomic region of interest with flanking transposase-binding sites, followed by in vitro transposase loading and introduction into the cells. The marker-driven approach involves cloning an open reading frame (ORF of interest into a vector containing a marker for mycoplasma transformation, as well as sites for transposase loading and random genomic integration. An innovative feature of this construct is to use a single promoter to express the transformation marker and the introduced ORF. The marker-driven approach can be conveniently applied to any exogenous or synthetic gene without any information on the effect of the gene on the strain, whereas the marker-less approach requires that the fragment has a recognizable effect. Using the marker-less method, we found that a region containing the nusG gene rescues a slow growth phenotype of a strain containing a larger deletion encompassing this gene. Using the marker-driven approach, we better defined this finding, thereby establishing that nusG is required for a normal growth rate in synthetic M. mycoides. These methods are suitable for complementation tests to identify genes responsible for assorted functions lacking in deletion mutants. These approaches are also expected to facilitate rapid testing of various natural and engineered genes or gene clusters from numerous sources in
Rajendra, Yashas; Balasubramanian, Sowmya; Peery, Robert B; Swartling, James R; McCracken, Neil A; Norris, Dawn L; Frye, Christopher C; Barnard, Gavin C
Chinese hamster ovary (CHO) cells remain the most popular host for the production of biopharmaceutical drugs, particularly monoclonal antibodies (mAbs), bispecific antibodies, and Fc-fusion proteins. Creating and characterizing the stable CHO clonally-derived cell lines (CDCLs) needed to manufacture these therapeutic proteins is a lengthy and laborious process. Therefore, CHO pools have increasingly been used to rapidly produce protein to support and enable preclinical drug development. We recently described the generation of CHO pools yielding mAb titers as high as 7.6 g/L in a 16 day bioprocess using piggyBac transposon-mediated gene integration. In this study, we wanted to understand why the piggyBac pool titers were significantly higher (2-10 fold) than the control CHO pools. Higher titers were the result of a combination of increased average gene copy number, significantly higher messenger RNA levels and the homogeneity (i.e. less diverse population distribution) of the piggyBac pools, relative to the control pools. In order to validate the use of piggyBac pools to support preclinical drug development, we then performed an in-depth product quality analysis of purified protein. The product quality of protein obtained from the piggyBac pools was very similar to the product quality profile of protein obtained from the control pools. Finally, we demonstrated the scalability of these pools from shake flasks to 36L bioreactors. Overall, these results suggest that gram quantities of therapeutic protein can be rapidly obtained from piggyBac CHO pools without significantly changing product quality attributes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:534-540, 2017. © 2017 American Institute of Chemical Engineers.
Sean F Landrette
Full Text Available Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.
Full Text Available Abstract Background Achromobacter sp. AO22 (formerly Alcaligenes sp. AO22, a bacterial strain isolated from a lead-contaminated industrial site in Australia, was previously found to be resistant to moderate to high levels of mercury, copper and other heavy metals. However, the nature and location of the genetic basis for mercuric ion resistance in this strain, had not been previously identified. Findings Achromobacter sp. AO22 contains a functional mer operon with all four essential genes (merRTPA and shows >99% DNA sequence identity to that of Tn501. The mer operon was present on a transposon, designated TnAO22, captured by introducing a broad-host-range IncP plasmid into Achromobacter sp. AO22 and subsequently transferring it to E. coli recipients. The transposition frequency of TnAO22 was 10-2 to 10-3 per target plasmid transferred. Analysis of TnAO22 sequence revealed it belonged to the Tn21 subgroup of the Tn3 superfamily of transposons, with the transposition module having >99% identity with Tn5051 of a Pseudomonas putida strain isolated from a water sample in New York. Conclusion TnAO22 is thus a new variant of Tn5051 of the Tn3 superfamily and the transposon and its associated mercury resistance system are among the few such systems reported in a soil bacterium. Achromobacter sp. AO22 can thus be exploited for applications such as in situ mercury bioremediation of contaminated sites, or the mobile unit and mer operon could be mobilized to other bacteria for similar purposes.
Oumeraci, Tonio; Jensen, Vanessa; Talbot, Steven R; Hofmann, Winfried; Kostrzewa, Markus; Schlegelberger, Brigitte; von Neuhoff, Nils; Häussler, Susanne
Pseudomonas aeruginosa is a gram-negative bacterium that is ubiquitously present in the aerobic biosphere. As an antibiotic-resistant facultative pathogen, it is a major cause of hospital-acquired infections. Its rapid and accurate identification is crucial in clinical and therapeutic environments. In a large-scale MALDI-TOF mass spectrometry-based screen of the Harvard transposon insertion mutant library of P. aeruginosa strain PA14, intact-cell proteome profile spectra of 5547 PA14 transposon mutants exhibiting a plethora of different phenotypes were acquired and analyzed. Of all P. aeruginosa PA14 mutant profiles 99.7% were correctly identified as P. aeruginosa with the Biotyper software on the species level. On the strain level, 99.99% of the profiles were mapped to five different individual P. aeruginosa Biotyper database entries. A principal component analysis-based approach was used to determine the most important discriminatory mass features between these Biotyper groups. Although technical replicas were consistently categorized to specific Biotyper groups in 94.2% of the mutant profiles, biological replicas were not, indicating that the distinct proteotypes are affected by growth conditions. The PA14 mutant profile collection presented here constitutes the largest coherent P. aeruginosa MALDI-TOF spectral dataset publicly available today. Transposon insertions in thousands of different P. aeruginosa genes did not affect species identification from MALDI-TOF mass spectra, clearly demonstrating the robustness of the approach. However, the assignment of the individual spectra to sub-groups proved to be non-consistent in biological replicas, indicating that the differentiation between biotyper groups in this nosocomial pathogen is unassured.
Wu, Chunxiao; Wang, Shu
piggyBac, a highly active transposon in insect and mammalian cells, is a very useful tool in genome manipulation. A new piggyBac-like element (PLE), named PLE-wu, was identified from a mutant baculovirus cultured in sf9 insect cells. This new transposon is 2931 bp in length and encodes two active forms of transposase, a 708-amino acid-long transposase and a short 576-residue-long transposase translated from a downstream in-frame initiation codon. PLE-wu has asymmetric terminal structures, containing 6-bp inverted terminal repeats, 32-bp imperfect inverted and direct sub-terminal repeats. Similar to piggyBac, PLE-wu exhibits traceless excision activity in both insect and mammalian cells, restoring the original TTAA target sequence upon excision. It also retains the insertion activity in mammalian cells with a plasmid to chromosome transposition rate about 10-fold higher than random integration. Plasmid rescue assays revealed that the TTAA target sequence was duplicated at the junctions of the insertion site. Deletion of the terminal sequences including the sub-terminal repeats decreased the transposition activity of the 708-residue-long transposase, while the transposition activity of the short form of transposase was not affected. With its low sequence similarity to piggyBac, PLE-wu will contribute to the understanding the mechanism of PLE transposition, as well as design of new transposon systems with higher activity. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
Arduino, Sonia M; Quiroga, María Paula; Ramírez, María Soledad; Merkier, Andrea Karina; Errecalde, Laura; Di Martino, Ana; Smayevsky, Jorgelina; Kaufman, Sara; Centrón, Daniela
Multiple transposons, integrons and carbapenemases were found in Klebsiella pneumoniae colistin-resistant isolates as well as a genomic resistance island of the AbaR type in Acinetobacter baumannii colistin-resistant isolates from different hospitals from Buenos Aires City. PFGE analysis showed a polyclonal dissemination of antimicrobial resistance mechanisms among K. pneumoniae isolates, while in A. baumannii isolates the epidemic clone 1 from South America was found. Resistance determinants associated with horizontal gene transfer are contributing to the evolution to pandrug resistance in both epidemic and sporadic clones.
Markljung, Ellen; Jiang, Lin; Jaffe, Jacob D
and find that the protein, named ZBED6, is previously unknown, specific for placental mammals, and derived from an exapted DNA transposon. Silencing of Zbed6 in mouse C2C12 myoblasts affected Igf2 expression, cell proliferation, wound healing, and myotube formation. Chromatin immunoprecipitation (Ch......, including development and transcriptional regulation. The phenotypic effects in mutant pigs and ZBED6-silenced C2C12 myoblasts, the extreme sequence conservation, its nucleolar localization, the broad tissue distribution, and the many target genes with essential biological functions suggest that ZBED6...... is an important transcription factor in placental mammals, affecting development, cell proliferation, and growth....
Sarver Aaron L
Full Text Available Abstract Background Next generation sequencing approaches applied to the analyses of transposon insertion junction fragments generated in high throughput forward genetic screens has created the need for clear informatics and statistical approaches to deal with the massive amount of data currently being generated. Previous approaches utilized to 1 map junction fragments within the genome and 2 identify Common Insertion Sites (CISs within the genome are not practical due to the volume of data generated by current sequencing technologies. Previous approaches applied to this problem also required significant manual annotation. Results We describe Transposon Annotation Poisson Distribution Association Network Connectivity Environment (TAPDANCE software, which automates the identification of CISs within transposon junction fragment insertion data. Starting with barcoded sequence data, the software identifies and trims sequences and maps putative genomic sequence to a reference genome using the bowtie short read mapper. Poisson distribution statistics are then applied to assess and rank genomic regions showing significant enrichment for transposon insertion. Novel methods of counting insertions are used to ensure that the results presented have the expected characteristics of informative CISs. A persistent mySQL database is generated and utilized to keep track of sequences, mappings and common insertion sites. Additionally, associations between phenotypes and CISs are also identified using Fisher’s exact test with multiple testing correction. In a case study using previously published data we show that the TAPDANCE software identifies CISs as previously described, prioritizes them based on p-value, allows holistic visualization of the data within genome browser software and identifies relationships present in the structure of the data. Conclusions The TAPDANCE process is fully automated, performs similarly to previous labor intensive approaches
Mason-Gamer Roberta J
Full Text Available Abstract Background Stowaway elements are short, non-autonomous DNA transposons categorized as miniature inverted-repeat transposable elements (MITEs. The high MITE copy number in grass genomes suggests an active history of amplification and insertion, but ongoing MITE activity has only rarely been seen, and ongoing Stowaway activity has never been observed. Thus, a phylogenetic perspective on presence vs. absence of elements in an aligned data set can provide valuable historical insights into the dynamics of MITE acquisition and loss. Results A Stowaway-like element resides within the fourth intron of a β-amylase gene in representatives of five genera in the wheat tribe, Triticeae. Its presence vs. absence was examined with reference to the β-amylase gene tree topology, and in light of sequence comparisons of the β-amylase elements to Triticeae Stowaway elements in the Entrez nucleotide database. Among the sequences lacking the element, there are five distinct putative excision footprints (one widespread and four restricted to unrelated lineages and two flanking deletions. The sequences that do contain elements are polyphyletic on the β-amylase tree, and their elements are divergent at the sequence level. The β-amylase elements do not form a monophyletic group relative to other Stowaway elements in Entrez; most are more similar to elements from other loci in other Triticeae genomes than they are to one another. Conclusion Combined, the phylogenetic distribution, sequence variation, and Entrez database comparisons indicate that a Stowaway-like element has undergone multiple deletions from and insertions into the same site in β-amylase intron 4 during the history of the tribe. The elements currently at the site represent multiple, distinct lineages that transcend generic boundaries. While patterns of Stowaway polymorphism across a phylogenetic data set do not allow evolutionary mechanisms to be inferred with certainty, they do provide
Schukkink, R F; Plasterk, R H
Tc1 is a transposon present in several copies in the genome of all natural isolates of the nematode C.elegans; it is actively transposing in many strains. In those strains Tc1 insertion is the main cause of spontaneous mutations. The transposon contains one large ORF that we call TcA; we assume that the TcA protein is the transposase of Tc1. We expressed TcA in E.coli, purified the protein and showed that it has a strong affinity for DNA (both single stranded and double stranded). A fusion pr...
Dubois, Emeline; Bischerour, Julien; Marmignon, Antoine; Mathy, Nathalie; Régnier, Vinciane; Bétermier, Mireille
Sequences related to transposons constitute a large fraction of extant genomes, but insertions within coding sequences have generally not been tolerated during evolution. Thanks to their unique nuclear dimorphism and to their original mechanism of programmed DNA elimination from their somatic nucleus (macronucleus), ciliates are emerging model organisms for the study of the impact of transposable elements on genomes. The germline genome of the ciliate Paramecium, located in its micronucleus, contains thousands of short intervening sequences, the IESs, which interrupt 47% of genes. Recent data provided support to the hypothesis that an evolutionary link exists between Paramecium IESs and Tc1/mariner transposons. During development of the macronucleus, IESs are excised precisely thanks to the coordinated action of PiggyMac, a domesticated piggyBac transposase, and of the NHEJ double-strand break repair pathway. A PiggyMac homolog is also required for developmentally programmed DNA elimination in another ciliate, Tetrahymena. Here, we present an overview of the life cycle of these unicellular eukaryotes and of the developmentally programmed genome rearrangements that take place at each sexual cycle. We discuss how ancient domestication of a piggyBac transposase might have allowed Tc1/mariner elements to spread throughout the germline genome of Paramecium, without strong counterselection against insertion within genes. PMID:22888464
Matsui, Toru; Nishino, Tomohiko
Analytical conditions using chromo azurol S was validated for quantification of siderophore in aqueous samples, followed by the characterization of siderophore derived from newly isolated moderately halophilic bacteria. Conditions with good linearity between the absorbance and the siderophore concentration were obtained at a siderophore concentration less than 20 µM, in the wavelength range between 630 and 660 nm with developing time for at least 2 h. Of the halophilic bacteria isolated from Tunisian soil, Halomonas sp., namely strain 21a was selected as siderophore producing halophiles. The strain produced siderophore significantly in the absence of iron in minimal medium. Siderophore-deficient mutant, namely IIa10, of the strain 21a was obtained from gene disruptant library constructed using transposon complex by electroporation. Genomic sequence analysis of the mutant IIa10 revealed that the transposon-inserted gene was TonB-dependent receptor. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Migicovsky, Zoe; Kovalchuk, Igor
Stress has a negative impact on crop yield by altering a gain in biomass and affecting seed set. Recent reports suggest that exposure to stress also influences the response of the progeny. In this paper, we analyzed seed size, leaf size, bolting time and transposon expression in 2 consecutive generations of Arabidopsis thaliana plants exposed to moderate UV-C stress. Since previous reports suggested a potential role of Dicer-like (DCL) proteins in the establishment of transgenerational response, we used dcl2, dcl3 and dcl4 mutants in parallel with wild-type plants. These studies revealed that leaf number decreased in the progeny of UV-C stressed plants, and bolting occurred later. Transposons were also re-activated in the progeny of stressed plants. Changes in the dcl mutants were less prominent than in wild-type plants. DCL2 and DCL3 appeared to be more important in the transgenerational stress memory than DCL4 because transgenerational changes were less profound in the dcl2 and dcl3 mutants.
Youvan, D C [Univ. of California, Berkeley; Elder, J T; Sandlin, D E; Zsebo, K; Alder, D P; Panopoulos, N J; Marrs, B L; Hearst, J E
Site-directed mutagenesis of the photosynthetic apparatus (PSA) genes in Rhodopseudomonas capsulata is presented utilizing a transposon Tn7 mutagenized R-prime. The R-prime, pRPS404, bears most of the genes necessary for the differentiation of the photosynthetic apparatus. Mutagenesis of the R-prime with Tn7 in Escherichia coli, conjugation into R. capsulata, and homologous recombination with the wild-type alleles efficiently generates photosynthetic apparatus lesions. Wild-type alleles are lost spontaneously and the Tn7-induced lesions are revealed by subsequent intramolecular recombination between IS21 insertion elements that bracket the prime sequences in direct repeat. The molecular nature of the intermediates involved in the transposition, recombination and deletion have been investigated by Southern hybridization analysis. The spontaneous loss of wild-type alleles after homologous recombination with the chromosome may be of general use to other prokaryotic site-directed transposon mutagenesis schemes. The IS21-mediated deletion of the prime DNA is dependent on the RecA protein in E. coli, generating the parental R-factor bearing one IS21 element. A genetic-physical map exists for a portion of the prime photosynthetic apparatus DNA. When Tn7 is inserted into a bacteriochlorophyll gene in the R-prime and then crossed into R. capsulata, mutants are produced that accumulate a bacteriochlorophyll precursor, which is in excellent agreement with the existing genetic-physical map. This corroborates the mutagenesis scheme.
Sun, Ting; Renner, Susanne S.; Xu, Yuxing; Qin, Yan; Wu, Jianqiang; Sun, Guiling
A growing body of evidence is pointing to an important role of horizontal gene transfer (HGT) in the evolution of higher plants. However, reports of HGTs of transposable elements (TEs) in plants are still scarce, and only one case is known of a class II transposon horizontally transferred between grasses. To investigate possible TE transfers in dicots, we performed transcriptome screening in the obligate root parasite Phelipanche aegyptiaca (Orobanchaceae), data-mining in the draft genome assemblies of four other Orobanchaceae, gene cloning, gene annotation in species with genomic information, and a molecular phylogenetic analysis. We discovered that the broomrape genera Phelipanche and Orobanche acquired two related nuclear genes (christened BO transposase genes), a new group of the hAT superfamily of class II transposons, from Asian Sisymbrieae or a closely related tribe of Brassicaceae, by HGT. The collinearity of the flanking genes, lack of a classic border structure, and low expression levels suggest that BO transposase genes cannot transpose in Brassicaceae, whereas they are highly expressed in P. aegyptiaca. PMID:27452947
Full Text Available Adoptive T cell therapy using chimeric antigen receptor (CAR-modified T cells is a promising cancer immunotherapy. We previously developed a non-viral method of gene transfer into T cells using a piggyBac transposon system to improve the cost-effectiveness of CAR-T cell therapy. Here, we have further improved our technology by a novel culture strategy to increase the transfection efficiency and to reduce the time of T cell manufacturing. Using a CH2CH3-free CD19-specific CAR transposon vector and combining irradiated activated T cells (ATCs as feeder cells and virus-specific T cell receptor (TCR stimulation, we achieved 51.4% ± 14% CAR+ T cells and 2.8-fold expansion after 14 culture days. Expanded CD19.CAR-T cells maintained a significant fraction of CD45RA+CCR7+ T cells and demonstrated potent antitumor activity against CD19+ leukemic cells both in vitro and in vivo. Therefore, piggyBac-based gene transfer may provide an alternative to viral gene transfer for CAR-T cell therapy.
Abdjad Asih Nawangsih
Full Text Available Xanthomonas oryzae pv oryzae the Causal Agent of Bacterial Leaf Blight of rice: Isolation, Characterization, and Study of Transposon Mutagenesis. X. oryzae pv. oryzae (Xoo causes bacterial leaf blight (BLB of rice (Oryza sativa L., a major disease that constrains production of the staple crop in many countries of the world. Identification of X. oryzae pv. oryzae (Xoo was conducted based on the disease symptoms, pathogenicity, morphological, physiological, and genetic characteristics of bacterial cultures isolated from the infected plants. Fifty bacterial isolates predicted as Xoo have been successfully isolated. They are aerobic, rod shaped, and Gram negative bacteria. The isolates were evaluated for their hypersensitivity in tobacco and pathogenicity in rice plant. Fifty isolates induced hypersensitive reaction in tobacco and showed pathogenicity symptom in rice in different length. Based on physiological test, hypersensitivity and pathogenicity reactions, three bacterial isolates strongly predicted as Xoo, i.e. STG21, STG42, and STG46, were non indole formation, non pigment fluorescent, hydrolyzed casein, catalase activity positive, but negative oxidase. Partial sequencing of 16S rRNA genes of STG21 and STG42 showed 80% and 82% homology with X. oryzae, respectively, while STG46 showed 84% homology with X. campestris. Mini-Tn5 transposon mutagenesis of STG21 generated one of the mutants (M5 lossed it’s ability to induce hypersensitive reaction in tobacco plant and deficient in pathogenicity on rice. The lesion length of rice leaf caused by the mutant M5 decreased up to 80%.
Cardinal, Marie-Josée; Kaur, Rajvinder; Singh, Jaswinder
Domestication and intensive selective breeding of plants has triggered erosion of genetic diversity of important stress-related alleles. Researchers highlight the potential of using wild accessions as a gene source for improvement of cereals such as barley, which has major economic and social importance worldwide. Previously, we have successfully introduced the maize Ac/Ds transposon system for gene identification in cultivated barley. The objective of current research was to investigate the response of Hordeum vulgare ssp. spontaneum wild barley accessions in tissue culture to standardize parameters for introduction of Ac/Ds transposons through genetic transformation. We investigated the response of ten wild barley genotypes for callus induction, regenerative green callus induction and regeneration of fertile plants. The activity of exogenous Ac/Ds elements was observed through a transient assay on immature wild barley embryos/callus whereby transformed embryos/calli were identified by the expression of GUS. Transient Ds expression bombardment experiments were performed on 352 pieces of callus (3-5 mm each) or immature embryos in 4 genotypes of wild barley. The transformation frequency of putative transgenic callus lines based on transient GUS expression ranged between 72 and100 % in wild barley genotypes. This is the first report of a transformation system in H. vulgare ssp. spontaneum.
Anwar, Sumadi Lukman; Wulaningsih, Wahyu; Lehmann, Ulrich
Transposable elements (TEs) comprise nearly half of the human genome and play an essential role in the maintenance of genomic stability, chromosomal architecture, and transcriptional regulation. TEs are repetitive sequences consisting of RNA transposons, DNA transposons, and endogenous retroviruses that can invade the human genome with a substantial contribution in human evolution and genomic diversity. TEs are therefore firmly regulated from early embryonic development and during the entire course of human life by epigenetic mechanisms, in particular DNA methylation and histone modifications. The deregulation of TEs has been reported in some developmental diseases, as well as for different types of human cancers. To date, the role of TEs, the mechanisms underlying TE reactivation, and the interplay with DNA methylation in human cancers remain largely unexplained. We reviewed the loss of epigenetic regulation and subsequent genomic instability, chromosomal aberrations, transcriptional deregulation, oncogenic activation, and aberrations of non-coding RNAs as the potential mechanisms underlying TE deregulation in human cancers. PMID:28471386
Sumadi Lukman Anwar
Full Text Available Transposable elements (TEs comprise nearly half of the human genome and play an essential role in the maintenance of genomic stability, chromosomal architecture, and transcriptional regulation. TEs are repetitive sequences consisting of RNA transposons, DNA transposons, and endogenous retroviruses that can invade the human genome with a substantial contribution in human evolution and genomic diversity. TEs are therefore firmly regulated from early embryonic development and during the entire course of human life by epigenetic mechanisms, in particular DNA methylation and histone modifications. The deregulation of TEs has been reported in some developmental diseases, as well as for different types of human cancers. To date, the role of TEs, the mechanisms underlying TE reactivation, and the interplay with DNA methylation in human cancers remain largely unexplained. We reviewed the loss of epigenetic regulation and subsequent genomic instability, chromosomal aberrations, transcriptional deregulation, oncogenic activation, and aberrations of non-coding RNAs as the potential mechanisms underlying TE deregulation in human cancers.
Aronovich, Elena L; Hyland, Kendra A; Hall, Bryan C; Bell, Jason B; Olson, Erik R; Rusten, Myra Urness; Hunter, David W; Ellinwood, N Matthew; McIvor, R Scott; Hackett, Perry B
The non-viral, integrating Sleeping Beauty (SB) transposon system is efficient in treating systemic monogenic disease in mice, including hemophilia A and B caused by deficiency of blood clotting factors and mucopolysaccharidosis types I and VII caused by α-L-iduronidase (IDUA) and β-glucuronidase (GUSB) deficiency, respectively. Modified approaches of the hydrodynamics-based procedure to deliver transposons to the liver in dogs were recently reported. Using the transgenic canine reporter secreted alkaline phosphatase (cSEAP), transgenic protein in the plasma was demonstrated for up to 6 weeks post infusion. This study reports that immunosuppression of dogs with gadolinium chloride (GdCl 3 ) prolonged the presence of cSEAP in the circulation up to 5.5 months after a single vector infusion. Transgene expression declined gradually but appeared to stabilize after about 2 months at approximately fourfold baseline level. Durability of transgenic protein expression in the plasma was inversely associated with transient increase of liver enzymes alanine transaminase and aspartate transaminase in response to the plasmid delivery procedure, which suggests a deleterious effect of hepatocellular toxicity on transgene expression. GdCl 3 treatment was ineffective for repeat vector infusions. In parallel studies, dogs were infused with potentially therapeutic transposons. Activities of transgenic IDUA and GUSB in plasma peaked at 50-350% of wildtype, but in the absence of immunosuppression lasted only a few days. Transposition was detectable by excision assay only when the most efficient transposase, SB100X, was used. Dogs infused with transposons encoding canine clotting factor IX (cFIX) were treated with GdCl 3 and showed expression profiles similar to those in cSEAP-infused dogs, with expression peaking at 40% wt (2 μg/mL). It is concluded that GdCl 3 can support extended transgene expression after hydrodynamic introduction of SB transposons in dogs, but that alternative
Rep, Martijn; van der Does, H Charlotte; Cornelissen, Ben J C
The facultative pathogenic fungus Fusarium oxysporum is known to harbour many different transposable and/or repetitive elements. We have identified Drifter, a novel DNA transposon of the hAT family in F. oxysporum. It was found adjoining SIX1-H, a truncated homolog of the SIX1 avirulence gene in F. oxysporum f. sp. lycopersici. Absence of a target site duplication as well as the 5' part of SIX1-H suggests that transposition of Drifter into the ancestor of SIX1-H was followed by loss of a chromosomal segment through recombination between Drifters. F. oxysporum isolates belonging to various formae speciales harbour between 0 and 5 full-length copies of Drifter and/or one or more copies with an internal deletion. Transcription of Drifter is activated during starvation for carbon or nitrogen.
Rollefson, Janet B.; Levar, Caleb E.; Bond, Daniel R.
Electron transfer from cells to metals and electrodes by the Fe(III)-reducing anaerobe Geobacter sulfurreducens requires proper expression of redox proteins and attachment mechanisms to interface bacteria with surfaces and neighboring cells. We hypothesized that transposon mutagenesis would complement targeted knockout studies in Geobacter spp. and identify novel genes involved in this process. Escherichia coli mating strains and plasmids were used to develop a conjugation protocol and deliver mini-Himar transposons, creating a library of over 8,000 mutants that was anaerobically arrayed and screened for a range of phenotypes, including auxotrophy for amino acids, inability to reduce Fe(III) citrate, and attachment to surfaces. Following protocol validation, mutants with strong phenotypes were further characterized in a three-electrode system to simultaneously quantify attachment, biofilm development, and respiratory parameters, revealing mutants defective in Fe(III) reduction but unaffected in electron transfer to electrodes (such as an insertion in GSU1330, a putative metal export protein) or defective in electrode reduction but demonstrating wild-type biofilm formation (due to an insertion upstream of the NHL domain protein GSU2505). An insertion in a putative ATP-dependent transporter (GSU1501) eliminated electrode colonization but not Fe(III) citrate reduction. A more complex phenotype was demonstrated by a mutant containing an insertion in a transglutaminase domain protein (GSU3361), which suddenly ceased to respire when biofilms reached approximately 50% of the wild-type levels. As most insertions were not in cytochromes but rather in transporters, two-component signaling proteins, and proteins of unknown function, this collection illustrates how biofilm formation and electron transfer are separate but complementary phenotypes, controlled by multiple loci not commonly studied in Geobacter spp. PMID:19395486
Carter, Jared D; Pereira, Andy; Dickerman, Allan W; Veilleux, Richard E
Tomato (Solanum lycopersicum) is a model organism for Solanaceae in both molecular and agronomic research. This project utilized Agrobacterium tumefaciens transformation and the transposon-tagging construct Activator (Ac)/Dissociator (Ds)-ATag-Bar_gosGFP to produce activation-tagged and knockout mutants in the processing tomato cultivar M82. The construct carried hygromycin resistance (hyg), green fluorescent protein (GFP), and the transposase (TPase) of maize (Zea mays) Activator major transcript X054214.1 on the stable Ac element, along with a 35S enhancer tetramer and glufosinate herbicide resistance (BAR) on the mobile Ds-ATag element. An in vitro propagation strategy was used to produce a population of 25 T0 plants from a single transformed plant regenerated in tissue culture. A T1 population of 11,000 selfed and cv M82 backcrossed progeny was produced from the functional T0 line. This population was screened using glufosinate herbicide, hygromycin leaf painting, and multiplex polymerase chain reaction (PCR). Insertion sites of transposed Ds-ATag elements were identified through thermal asymmetric interlaced PCR, and resulting product sequences were aligned to the recently published tomato genome. A population of 509 independent, Ds-only transposant lines spanning all 12 tomato chromosomes has been developed. Insertion site analysis demonstrated that more than 80% of these lines harbored Ds insertions conducive to activation tagging. The capacity of the Ds-ATag element to alter transcription was verified by quantitative real-time reverse transcription-PCR in two mutant lines. The transposon-tagged lines have been immortalized in seed stocks and can be accessed through an online database, providing a unique resource for tomato breeding and analysis of gene function in the background of a commercial tomato cultivar.
Loftus, Brendan J.; Fung, Eula; Roncaglia, Paola; Rowley, Don; Amedeo, Paolo; Bruno, Dan; Vamathevan, Jessica; Miranda, Molly; Anderson, Iain J.; Fraser, James A.; Allen, Jonathan E.; Bosdet, Ian E.; Brent, Michael R.; Chiu, Readman; Doering, Tamara L.; Donlin, Maureen J.; D’Souza, Cletus A.; Fox, Deborah S.; Grinberg, Viktoriya; Fu, Jianmin; Fukushima, Marilyn; Haas, Brian J.; Huang, James C.; Janbon, Guilhem; Jones, Steven J. M.; Koo, Hean L.; Krzywinski, Martin I.; Kwon-Chung, June K.; Lengeler, Klaus B.; Maiti, Rama; Marra, Marco A.; Marra, Robert E.; Mathewson, Carrie A.; Mitchell, Thomas G.; Pertea, Mihaela; Riggs, Florenta R.; Salzberg, Steven L.; Schein, Jacqueline E.; Shvartsbeyn, Alla; Shin, Heesun; Shumway, Martin; Specht, Charles A.; Suh, Bernard B.; Tenney, Aaron; Utterback, Terry R.; Wickes, Brian L.; Wortman, Jennifer R.; Wye, Natasja H.; Kronstad, James W.; Lodge, Jennifer K.; Heitman, Joseph; Davis, Ronald W.; Fraser, Claire M.; Hyman, Richard W.
Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its ~20-megabase genome, which contains ~6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties, and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes. PMID:15653466
Full Text Available RNA silencing is a defense system against "genomic parasites" such as transposable elements (TE, which are potentially harmful to host genomes. In plants, transcripts from TEs induce production of double-stranded RNAs (dsRNAs and are processed into small RNAs (small interfering RNAs, siRNAs that suppress TEs by RNA-directed DNA methylation. Thus, the majority of TEs are epigenetically silenced. On the other hand, most of the eukaryotic genome is composed of TEs and their remnants, suggesting that TEs have evolved countermeasures against host-mediated silencing. Under some circumstances, TEs can become active and increase in copy number. Knowledge is accumulating on the mechanisms of TE silencing by the host; however, the mechanisms by which TEs counteract silencing are poorly understood. Here, we show that a class of TEs in rice produces a microRNA (miRNA to suppress host silencing. Members of the microRNA820 (miR820 gene family are located within CACTA DNA transposons in rice and target a de novo DNA methyltransferase gene, OsDRM2, one of the components of epigenetic silencing. We confirmed that miR820 negatively regulates the expression of OsDRM2. In addition, we found that expression levels of various TEs are increased quite sensitively in response to decreased OsDRM2 expression and DNA methylation at TE loci. Furthermore, we found that the nucleotide sequence of miR820 and its recognition site within the target gene in some Oryza species have co-evolved to maintain their base-pairing ability. The co-evolution of these sequences provides evidence for the functionality of this regulation. Our results demonstrate how parasitic elements in the genome escape the host's defense machinery. Furthermore, our analysis of the regulation of OsDRM2 by miR820 sheds light on the action of transposon-derived small RNAs, not only as a defense mechanism for host genomes but also as a regulator of interactions between hosts and their parasitic elements.
Berendsen, Erwin M.; Koning, Rosella A.; Boekhorst, Jos; de Jong, Anne; Kuipers, Oscar P.; Wells-Bennik, Marjon H. J.
Bacterial endospore formers can produce spores that are resistant to many food processing conditions, including heat. Some spores may survive heating processes aimed at production of commercially sterile foods. Recently, it was shown that a spoVA operon, designated spoVA2mob, present on a Tn1546 transposon in Bacillus subtilis, leads to profoundly increased wet heat resistance of B. subtilis spores. Such Tn1546 transposon elements including the spoVA2mob operon were also found in several strains of Bacillus amyloliquefaciens and Bacillus licheniformis, and these strains were shown to produce spores with significantly higher resistances to wet heat than their counterparts lacking this transposon. In this study, the locations and compositions of Tn1546 transposons encompassing the spoVA2mob operons in B. amyloliquefaciens and B. licheniformis were analyzed. Introduction of these spoVA2mob operons into B. subtilis 168 (producing spores that are not highly heat resistant) rendered mutant 168 strains that produced high-level heat resistant spores, demonstrating that these elements in B. amyloliquefaciens and B. licheniformis are responsible for high level heat resistance of spores. Assessment of growth of the nine strains of each species between 5.2°C and 57.7°C showed some differences between strains, especially at lower temperatures, but all strains were able to grow at 57.7°C. Strains of B. amyloliquefaciens and B. licheniformis that contain the Tn1546 elements (and produce high-level heat resistant spores) grew at temperatures similar to those of their Tn1546-negative counterparts that produce low-level heat resistant spores. The findings presented in this study allow for detection of B. amyloliquefaciens and B. licheniformis strains that produce highly heat resistant spores in the food chain. PMID:27994575
Background In the silkworm, Bombyx mori, femaleness is strongly controlled by the female-specific W chromosome. Originally, it was presumed that the W chromosome encodes female-determining gene(s), accordingly called Fem. However, to date, neither Fem nor any protein-coding gene has been identified from the W chromosome. Instead, the W chromosome is occupied with numerous transposon-related sequences. Interestingly, the silkworm W chromosome is a source of female-enriched PIWI-interacting RNAs (piRNAs). piRNAs are small RNAs of 23-30 nucleotides in length, which are required for controlling transposon activity in animal gonads. A recent study has identified a novel mutant silkworm line called KG, whose mutation in the W chromosome causes severe female masculinization. However, the molecular nature of KG line has not been well characterized yet. Results Here we molecularly characterize the KG line. Genomic PCR analyses using currently available W chromosome-specific PCR markers indicated that no large deletion existed in the KG W chromosome. Genetic analyses demonstrated that sib-crosses within the KG line suppressed masculinization. Masculinization reactivated when crossing KG females with wild type males. Importantly, the KG ovaries exhibited a significantly abnormal transcriptome. First, the KG ovaries misexpressed testis-specific genes. Second, a set of female-enriched piRNAs was downregulated in the KG ovaries. Third, several transposons were overexpressed in the KG ovaries. Conclusions Collectively, the mutation in the KG W chromosome causes broadly altered expression of testis-specific genes, piRNAs, and transposons. To our knowledge, this is the first study that describes a W chromosome mutant with such an intriguing phenotype. PMID:22452797
Bouuaert, Corentin Claeys; Tellier, Michael; Chalmers, Ronald
The development of transposon-based genome manipulation tools can benefit greatly from understanding transposons’ inherent regulatory mechanisms. The Tc1-mariner transposons, which are being widely used in biotechnological applications, are subject to a self-inhibitory mechanism whereby increasing transposase expression beyond a certain point decreases the rate of transposition. In a recent paper, Liu and Chalmers performed saturating mutagenesis on the highly conserved WVPHEL motif in the mariner-family transposase from the Hsmar1 element. Curiously, they found that the majority of all possible single mutations were hyperactive. Biochemical characterizations of the mutants revealed that the hyperactivity is due to a defect in communication between transposase subunits, which normally regulates transposition by reducing the rate of synapsis. This provides important clues for improving transposon-based tools. However, some WVPHEL mutants also showed features that would be undesirable for most biotechnological applications: they showed uncontrolled DNA cleavage activities and defects in the coordination of cleavage between the two transposon ends. The study illustrates how the knowledge of inhibitory mechanisms can help improve transposon tools but also highlights an important challenge, which is to specifically target a regulatory mechanism without affecting other important functions of the transposase. PMID:24812590
Macias, Vanessa M; Jimenez, Alyssa J; Burini-Kojin, Bianca; Pledger, David; Jasinskiene, Nijole; Phong, Celine Hien; Chu, Karen; Fazekas, Aniko; Martin, Kelcie; Marinotti, Osvaldo; James, Anthony A
Transposons are a class of selfish DNA elements that can mobilize within a genome. If mobilization is accompanied by an increase in copy number (replicative transposition), the transposon may sweep through a population until it is fixed in all of its interbreeding members. This introgression has been proposed as the basis for drive systems to move genes with desirable phenotypes into target species. One such application would be to use them to move a gene conferring resistance to malaria parasites throughout a population of vector mosquitos. We assessed the feasibility of using the piggyBac transposon as a gene-drive mechanism to distribute anti-malarial transgenes in populations of the malaria vector, Anopheles stephensi. We designed synthetic gene constructs that express the piggyBac transposase in the female germline using the control DNA of the An. stephensi nanos orthologous gene linked to marker genes to monitor inheritance. Two remobilization events were observed with a frequency of one every 23 generations, a rate far below what would be useful to drive anti-pathogen transgenes into wild mosquito populations. We discuss the possibility of optimizing this system and the impetus to do so. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.
Weerdenburg, Eveline M.
The interaction of environmental bacteria with unicellular eukaryotes is generally considered a major driving force for the evolution of intracellular pathogens, allowing them to survive and replicate in phagocytic cells of vertebrate hosts. To test this hypothesis on a genome-wide level, we determined for the intracellular pathogen Mycobacterium marinum whether it uses conserved strategies to exploit host cells from both protozoan and vertebrate origin. Using transposon-directed insertion site sequencing (TraDIS), we determined differences in genetic requirements for survival and replication in phagocytic cells of organisms from different kingdoms. In line with the general hypothesis, we identified a number of general virulence mechanisms, including the type VII protein secretion system ESX-1, biosynthesis of polyketide lipids, and utilization of sterols. However, we were also able to show that M. marinum contains an even larger set of host-specific virulence determinants, including proteins involved in the modification of surface glycolipids and, surprisingly, the auxiliary proteins of the ESX-1 system. Several of these factors were in fact counterproductive in other hosts. Therefore, M. marinum contains different sets of virulence factors that are tailored for specific hosts. Our data imply that although amoebae could function as a training ground for intracellular pathogens, they do not fully prepare pathogens for crossing species barriers.
Full Text Available Pigment epithelium derived factor (PEDF is a potent antiangiogenic, neurotrophic, and neuroprotective molecule that is the endogenous inhibitor of vascular endothelial growth factor (VEGF in the retina. An ex vivo gene therapy approach based on transgenic overexpression of PEDF in the eye is assumed to rebalance the angiogenic-antiangiogenic milieu of the retina, resulting in growth regression of choroidal blood vessels, the hallmark of neovascular age-related macular degeneration. Here, we show that rat pigment epithelial cells can be efficiently transfected with the PEDF-expressing non-viral hyperactive Sleeping Beauty transposon system delivered in a form free of antibiotic resistance marker miniplasmids. The engineered retinal and iris pigment epithelium cells secrete high (141 ± 13 and 222 ± 14 ng PEDF levels in 72 hr in vitro. In vivo studies showed cell survival and insert expression during at least 4 months. Transplantation of the engineered cells to the subretinal space of a rat model of choroidal neovascularization reduces almost 50% of the development of new vessels.
Kiljunen, Saija; Pajunen, Maria I; Dilks, Kieran; Storf, Stefanie; Pohlschroder, Mechthild; Savilahti, Harri
Archaea share fundamental properties with bacteria and eukaryotes. Yet, they also possess unique attributes, which largely remain poorly characterized. Haloferax volcanii is an aerobic, moderately halophilic archaeon that can be grown in defined media. It serves as an excellent archaeal model organism to study the molecular mechanisms of biological processes and cellular responses to changes in the environment. Studies on haloarchaea have been impeded by the lack of efficient genetic screens that would facilitate the identification of protein functions and respective metabolic pathways. Here, we devised an insertion mutagenesis strategy that combined Mu in vitro DNA transposition and homologous-recombination-based gene targeting in H. volcanii. We generated an insertion mutant library, in which the clones contained a single genomic insertion. From the library, we isolated pigmentation-defective and auxotrophic mutants, and the respective insertions pinpointed a number of genes previously known to be involved in carotenoid and amino acid biosynthesis pathways, thus validating the performance of the methodologies used. We also identified mutants that had a transposon insertion in a gene encoding a protein of unknown or putative function, demonstrating that novel roles for non-annotated genes could be assigned. We have generated, for the first time, a random genomic insertion mutant library for a halophilic archaeon and used it for efficient gene discovery. The library will facilitate the identification of non-essential genes behind any specific biochemical pathway. It represents a significant step towards achieving a more complete understanding of the unique characteristics of halophilic archaea.
Transposable elements (TEs) are capable of mobilizing from one genomic location to other, with changes in their copy numbers. Mutator-like elements (MULEs) are DNA transposons characterized by 9 bp target site duplications (TSDs), with high variability in sequence and length, and include non-conserved terminal inverted repeats (TIRs). We identified and characterized a family of Mutator-like elements designated as Shahroz. The structural and molecular analyses revealed that family had a small number of mostly defective non-autonomous MULEs and has shown limited activity in the evolutionary history of the Brassica A-genome. The Shahroz elements range in size from 2734 to 3160 bp including 76 bp imperfect TIRs and 9 bp variable TSDs. The individual copies have shown high homology (52-99%) in their entire lengths. The study revealed that the elements are less in numbers but active in Brassica rapa genomes and PCR amplification revealed their specificity and amplification in A-genome containing diploid and polyploids Brassica. The phylogenetic analysis of Brassica MULEs with other plant Mutator elements revealed that no correlation exists between Brassica MULEs and other elements suggesting a separate line of evolution. Analyzing the regions flanking the insertions revealed that the insertions have showed a preference for AT rich regions. The detailed study of these insertions revealed that although less in number and small sizes, they have played a role in Brassica genome evolution by their mobilization. (author)
Full Text Available The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms around plant cells. If the pathogen can suppress the plant's natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction.
Lawrence Mark L
Full Text Available Abstract Background Salmonellosis may be a food safety problem when raw food products are mishandled and not fully cooked. In previous work, we developed bioluminescent Salmonella enterica serotypes using a plasmid-based reporting system that can be used for real-time monitoring of the pathogen's growth on food products in short term studies. In this study, we report the use of a Tn7-based transposon system for subcloning of luxCDABE genes into the chromosome of eleven Salmonella enterica serotypes isolated from the broiler production continuum. Results We found that the lux operon is constitutively expressed from the chromosome post-transposition and the lux cassette is stable without external pressure, i.e. antibiotic selection, for all Salmonella enterica serotypes used. Bioluminescence expression is based on an active electron transport chain and is directly related with metabolic activity. This relationship was quantified by measuring bioluminescence against a temperature gradient in aqueous solution using a luminometer. In addition, bioluminescent monitoring of two serotypes confirmed that our chicken skin model has the potential to be used to evaluate pathogen mitigation strategies. Conclusions This study demonstrated that our new stable reporting system eliminates bioluminescence variation due to plasmid instability and provides a reliable real-time experimental system to study application of preventive measures for Salmonella on food products in real-time for both short and long term studies.
Ferreira, Rafael Marini; de Oliveira, Amanda Carolina P; Moreira, Leandro M; Belasque, José; Gourbeyre, Edith; Siguier, Patricia; Ferro, Maria Inês T; Ferro, Jesus A; Chandler, Michael; Varani, Alessandro M
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. Xanthomonas genomes carry many insertion sequences (IS) and transposons, which play an important role in their evolution and architecture. This study reveals a key relationship between transposons and pathogenicity determinants in
Full Text Available Abstract Background Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. Results A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100-containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4-monophosphatases (EC 220.127.116.11. Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown
Gloux, Karine; Leclerc, Marion; Iliozer, Harout; L'Haridon, René; Manichanh, Chaysavanh; Corthier, Gérard; Nalin, Renaud; Blottière, Hervé M; Doré, Joël
Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.
Beiles, Avigdor; Raz, Shmuel; Ben-Abu, Yuval; Nevo, Eviatar
The current analysis of transposon elements (TE) in Drosophila melanogaster at Evolution Canyon, (EC), Israel, is based on data and analysis done by our collaborators (Drs. J. Gonzalez, J. Martinez and W. Makalowski, this issue). They estimated the frequencies of 28 TEs (transposon elements) in fruit flies (D. melanogaster) from the ecologically tropic, hot, and dry south-facing slope (SFS) or "African" slope (AS) of EC and compared it with the TE frequencies on the temperate-cool and humid north-facing slope (NFS) or "European" slope (ES), separated, on average, by 250 m. The flies were sampled from two stations on each slope. We received their results, including the frequencies of each TE on each slope, and the probabilities of the statistical analyses (G-tests) of each TE separately. We continued the analysis of the inter-slope differences of the frequencies of the TEs, and based our different conclusions on that analysis and on the difference between micro (=EC) and macro (2000 km.) comparisons [Gonzalez et al. 2015 doi: 10.1186/s13062-015-0075-4 ]. Our collaborators based all their conclusions on the non-significant results of each of the individual tests of the 28 TEs. We analysed also the distribution of the TE differences between the slopes, based on their results. Thirteen TEs were more frequent on the SFS, 11 were more frequent on the NFS, and four had equal frequencies. Because of the equalizing effect of the ongoing migration, only small and temporary differences between the slopes (0 - 0.06) were regarded by us as random fluctuations (drift). Three TEs were intermediate (0.08-0.09) and await additional research. The 11 TEs with large frequency differences (0.12 - 0.22) were regarded by us as putative adaptive TEs, because the equalizing power of ongoing migration will eliminate random large differences. Five of them were higher on the SFS and six were higher on the NFS. Gaps in the distribution of the differences distinguished between the large and
Marzo, Mar; Bello, Xabier; Puig, Marta; Maside, Xulio; Ruiz, Alfredo
Galileo is a transposable element responsible for the generation of three chromosomal inversions in natural populations of Drosophila buzzatii. Although the most characteristic feature of Galileo is the long internally-repetitive terminal inverted repeats (TIRs), which resemble the Drosophila Foldback element, its transposase-coding sequence has led to its classification as a member of the P-element superfamily (Class II, subclass 1, TIR order). Furthermore, Galileo has a wide distribution in the genus Drosophila, since it has been found in 6 of the 12 Drosophila sequenced genomes. Among these species, D. mojavensis, the one closest to D. buzzatii, presented the highest diversity in sequence and structure of Galileo elements. In the present work, we carried out a thorough search and annotation of all the Galileo copies present in the D. mojavensis sequenced genome. In our set of 170 Galileo copies we have detected 5 Galileo subfamilies (C, D, E, F, and X) with different structures ranging from nearly complete, to only 2 TIR or solo TIR copies. Finally, we have explored the structural and length variation of the Galileo copies that point out the relatively frequent rearrangements within and between Galileo elements. Different mechanisms responsible for these rearrangements are discussed. Although Galileo is a transposable element with an ancient history in the D. mojavensis genome, our data indicate a recent transpositional activity. Furthermore, the dynamism in sequence and structure, mainly affecting the TIRs, suggests an active exchange of sequences among the copies. This exchange could lead to new subfamilies of the transposon, which could be crucial for the long-term survival of the element in the genome.
Kim, Hyun-Soo [Ewha Womans Univ., Seoul (Korea, Republic of). Dept. of Life Science; Kim, Na-Rae [Ewha Womans Univ., Seoul (Korea, Republic of). Div. of Life and Pharmaceutical Sciences; Kim, Wankee [Ewha Womans Univ., Seoul (Korea, Republic of). Dept. of Life Science; Ajou Univ., Suwon (Korea, Republic of). Inst. for Medical Sciences; Choi, Wonja [Ewha Womans Univ., Seoul (Korea, Republic of). Dept. of Life Science; Ewha Womans Univ., Seoul (Korea, Republic of). Microbial Resources Research Center
Furfural is one of the major inhibitors generated during sugar production from cellulosic materials and, as an aldehyde, inhibits various cellular activities of microorganisms used, leading to prolonged lag time during ethanologenic fermentation. Since Saccharomyces cerevisiae strains tolerant to furfural are of great economic benefit in producing bioethanol, much effort to obtain more efficient strains continues to be made. In this study, we examined the furfural tolerance of transposon mutant strains (Tn 1-5) with enhanced ethanol tolerance and found that one of them (Tn 2), in which SSK2 is downregulated at the transcriptional level, displayed improved furfural tolerance. Such phenotype was abolished by complementation of the entire open reading frame of SSK2, which encodes a mitogen-activated protein (MAP) kinase kinase kinase of the high osmolarity glycerol (HOG) signaling pathway, suggesting an inhibitory effect of SSK2 in coping with furfural stress. Tn 2 showed a significant decrease in the intracellular level of reactive oxygen species (ROS) and early and high activation of Hog1p, a MAP kinase integral to the HOG pathway in response to furfural. The transcriptional levels of CTT1 and GLR1, two of known Hog1p downstream target genes whose protein products are involved in reducing ROS, were increased by 43 % and 56 % respectively compared with a control strain, probably resulting in the ROS decrease. Tn 2 also showed a shortened lag time during fermentation in the presence of furfural, resulting from efficient conversion of furfural to non-toxic (or less toxic) furfuryl alcohol. Taken together, the enhanced furfural tolerance of Tn 2 is suggested to be conferred by the combined effect of an early event of less ROS accumulation and a late event of efficient detoxification of furfural. (orig.)
Kim, Hyun-Soo; Kim, Na-Rae; Kim, Wankee; Choi, Wonja
Furfural is one of the major inhibitors generated during sugar production from cellulosic materials and, as an aldehyde, inhibits various cellular activities of microorganisms used, leading to prolonged lag time during ethanologenic fermentation. Since Saccharomyces cerevisiae strains tolerant to furfural are of great economic benefit in producing bioethanol, much effort to obtain more efficient strains continues to be made. In this study, we examined the furfural tolerance of transposon mutant strains (Tn 1-5) with enhanced ethanol tolerance and found that one of them (Tn 2), in which SSK2 is downregulated at the transcriptional level, displayed improved furfural tolerance. Such phenotype was abolished by complementation of the entire open reading frame of SSK2, which encodes a mitogen-activated protein (MAP) kinase kinase kinase of the high osmolarity glycerol (HOG) signaling pathway, suggesting an inhibitory effect of SSK2 in coping with furfural stress. Tn 2 showed a significant decrease in the intracellular level of reactive oxygen species (ROS) and early and high activation of Hog1p, a MAP kinase integral to the HOG pathway in response to furfural. The transcriptional levels of CTT1 and GLR1, two of known Hog1p downstream target genes whose protein products are involved in reducing ROS, were increased by 43 % and 56 % respectively compared with a control strain, probably resulting in the ROS decrease. Tn 2 also showed a shortened lag time during fermentation in the presence of furfural, resulting from efficient conversion of furfural to non-toxic (or less toxic) furfuryl alcohol. Taken together, the enhanced furfural tolerance of Tn 2 is suggested to be conferred by the combined effect of an early event of less ROS accumulation and a late event of efficient detoxification of furfural.
Deng, Ying; Nagachar, Nivedita; Xiao, Chaowen; Tien, Ming
The acs operon of Gluconacetobacter is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel−) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript “Ax” indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel− mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel− mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding β-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis. PMID:24013627
Full Text Available Miniature inverted-repeat transposable elements (MITEs are numerically predominant transposable elements in the rice genome, and their activities have influenced the evolution of genes. Very little is known about how MITEs can rapidly amplify to thousands in the genome. The rice MITE mPing is quiescent in most cultivars under natural growth conditions, although it is activated by various stresses, such as tissue culture, gamma-ray irradiation, and high hydrostatic pressure. Exceptionally in the temperate japonica rice strain EG4 (cultivar Gimbozu, mPing has reached over 1000 copies in the genome, and is amplifying owing to its active transposition even under natural growth conditions. Being the only active MITE, mPing in EG4 is an appropriate material to study how MITEs amplify in the genome. Here, we provide important findings regarding the transposition and amplification of mPing in EG4. Transposon display of mPing using various tissues of a single EG4 plant revealed that most de novo mPing insertions arise in embryogenesis during the period from 3 to 5 days after pollination (DAP, and a large majority of these insertions are transmissible to the next generation. Locus-specific PCR showed that mPing excisions and insertions arose at the same time (3 to 5 DAP. Moreover, expression analysis and in situ hybridization analysis revealed that Ping, an autonomous partner for mPing, was markedly up-regulated in the 3 DAP embryo of EG4, whereas such up-regulation of Ping was not observed in the mPing-inactive cultivar Nipponbare. These results demonstrate that the early embryogenesis-specific expression of Ping is responsible for the successful amplification of mPing in EG4. This study helps not only to elucidate the whole mechanism of mPing amplification but also to further understand the contribution of MITEs to genome evolution.
Watabe, Kazuyuki; Mimuro, Mamoru; Tsuchiya, Tohru
Synechocystis sp. PCC 6803 (Synechocystis) is the first sequenced photosynthetic organism and has two advantages: natural transformation and light-activated heterotrophic growth. Such characteristics have mainly promoted reverse genetic analysis in this organism, however, to date approximately 50% of genes are still annotated as 'unknown protein' or 'hypothetical protein'. Therefore, forward genetic analysis is required for the identification of significant genes responsible for photosynthesis and other physiological phenomena among the genes of unknown function. The in vivo transposon mutagenesis system is one of the major methods for random mutagenesis. However, present in vivo transposon mutagenesis systems for cyanobacteria face problems such as relatively low frequency of transposition and repeated transposition in the host cells. In this study, we constructed vectors based on a mini-Tn5-derived vector that was designed to prevent repeated transposition. Our vectors carry a hyperactive transposase and optimized recognition sequence of transposase, which were reported to enhance frequency of transposition. Using the vector, we succeeded in highly frequent transposition (9×10(-3) per recipient cell) in Synechocystis. Transposon insertion sites of 10 randomly selected mutants indicated that the insertion sites spread throughout the genome with low sequence dependency. Furthermore, one of the 10 mutants exhibited the slow-growing phenotype, and the mutant was functionally complemented by using our expression vector. Our system also worked with another model cyanobacterium, Synechococcus elongatus PCC 7942, with high frequency. These results indicate that the developed system can be applied to the forward genetic analysis of a broad range of cyanobacteria. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: email@example.com.
Kang, Yu; Zhang, Xiaoyan; Jiang, Wei; Wu, Chaoqun; Chen, Chunmei; Zheng, Yufang; Gu, Jianren; Xu, Congjian
Compared with viral vectors, nonviral vectors are less immunogenic, more stable, safer and easier to replication for application in cancer gene therapy. However, nonviral gene delivery system has not been extensively used because of the low transfection efficiency and the short transgene expression, especially in vivo. It is desirable to develop a nonviral gene delivery system that can support stable genomic integration and persistent gene expression in vivo. Here, we used a composite nonviral gene delivery system consisting of the piggyBac (PB) transposon and polyethylenimine (PEI) for long-term transgene expression in mouse ovarian tumors. A recombinant plasmid PB [Act-RFP, HSV-tk] encoding both the herpes simplex thymidine kinase (HSV-tk) and the monomeric red fluorescent protein (mRFP1) under PB transposon elements was constructed. This plasmid and the PBase plasmid were injected into ovarian cancer tumor xenografts in mice by in vivo PEI system. The antitumor effects of HSV-tk/ganciclovir (GCV) system were observed after intraperitoneal injection of GCV. Histological analysis and TUNEL assay were performed on the cryostat sections of the tumor tissue. Plasmid construction was confirmed by PCR analysis combined with restrictive enzyme digestion. mRFP1 expression could be visualized three weeks after the last transfection of pPB/TK under fluorescence microscopy. After GCV admission, the tumor volume of PB/TK group was significantly reduced and the tumor inhibitory rate was 81.96% contrasted against the 43.07% in the TK group. Histological analysis showed that there were extensive necrosis and lymphocytes infiltration in the tumor tissue of the PB/TK group but limited in the tissue of control group. TUNEL assays suggested that the transfected cells were undergoing apoptosis after GCV admission in vivo. Our results show that the nonviral gene delivery system coupling PB transposon with PEI can be used as an efficient tool for gene therapy in ovarian cancer
Genome-wide transposon mutagenesis of Proteus mirabilis: Essential genes, fitness factors for catheter-associated urinary tract infection, and the impact of polymicrobial infection on fitness requirements
Smith, Sara N.; Zhao, Lili; Wu, Weisheng
The Gram-negative bacterium Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Numerous prior studies have uncovered virulence factors for P. mirabilis pathogenicity in a murine model of ascending UTI, but little is known concerning pathogenesis during CAUTI or polymicrobial infection. In this study, we utilized five pools of 10,000 transposon mutants each and transposon insertion-site sequencing (Tn-Seq) to identify the full arsenal of P. mirabilis HI4320 fitness factors for single-species versus polymicrobial CAUTI with Providencia stuartii BE2467. 436 genes in the input pools lacked transposon insertions and were therefore concluded to be essential for P. mirabilis growth in rich medium. 629 genes were identified as P. mirabilis fitness factors during single-species CAUTI. Tn-Seq from coinfection with P. stuartii revealed 217/629 (35%) of the same genes as identified by single-species Tn-Seq, and 1353 additional factors that specifically contribute to colonization during coinfection. Mutants were constructed in eight genes of interest to validate the initial screen: 7/8 (88%) mutants exhibited the expected phenotypes for single-species CAUTI, and 3/3 (100%) validated the expected phenotypes for polymicrobial CAUTI. This approach provided validation of numerous previously described P. mirabilis fitness determinants from an ascending model of UTI, the discovery of novel fitness determinants specifically for CAUTI, and a stringent assessment of how polymicrobial infection influences fitness requirements. For instance, we describe a requirement for branched-chain amino acid biosynthesis by P. mirabilis during coinfection due to high-affinity import of leucine by P. stuartii. Further investigation of genes and pathways that provide a competitive advantage during both single-species and polymicrobial CAUTI will likely provide robust targets for therapeutic intervention to reduce P. mirabilis
Full Text Available Transposable elements are a serious threat for genome integrity and their control via small RNA mediated silencing pathways is an ancient strategy. The fruit fly Drosophila melanogaster has two silencing activities that target transposons: endogenous siRNAs (esiRNAs or endo-siRNAs and Piwi-interacting small RNAs (piRNAs. The biogenesis of endo-siRNAs involves the Dicer-2 co-factors Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2, which primarily helps to direct siRNAs into the RNA interference effector Ago2. Nonetheless, loss of either protein is not sufficient to produce a phenotype comparable with a dcr-2 mutation. We provide further deep sequencing evidence supporting the notion that R2D2 and Loqs-PD have partially overlapping function. Certain transposons display a preference for either dsRBD-protein during production or loading; this appeared to correlate neither with overall abundance, classification of the transposon or a specific site of genomic origin. The endo-siRNA biogenesis pathway in germline operates according to the same principles as the existing model for the soma, and its impairment does not significantly affect piRNAs. Expanding the analysis, we confirmed the occurrence of somatic piRNA-like RNAs (pilRNAs that show a ping-pong signature. We detected expression of the Piwi-family protein mRNAs only barely above background, indicating that the somatic pilRNAs may arise from a small sub-population of somatic cells that express a functional piRNA pathway.
Silje V Veiseth
Full Text Available Chromatin structure and gene expression are regulated by posttranslational modifications (PTMs on the N-terminal tails of histones. Mono-, di-, or trimethylation of lysine residues by histone lysine methyltransferases (HKMTases can have activating or repressive functions depending on the position and context of the modified lysine. In Arabidopsis, trimethylation of lysine 9 on histone H3 (H3K9me3 is mainly associated with euchromatin and transcribed genes, although low levels of this mark are also detected at transposons and repeat sequences. Besides the evolutionarily conserved SET domain which is responsible for enzyme activity, most HKMTases also contain additional domains which enable them to respond to other PTMs or cellular signals. Here we show that the N-terminal WIYLD domain of the Arabidopsis SUVR4 HKMTase binds ubiquitin and that the SUVR4 product specificity shifts from di- to trimethylation in the presence of free ubiquitin, enabling conversion of H3K9me1 to H3K9me3 in vitro. Chromatin immunoprecipitation and immunocytological analysis showed that SUVR4 in vivo specifically converts H3K9me1 to H3K9me3 at transposons and pseudogenes and has a locus-specific repressive effect on the expression of such elements. Bisulfite sequencing indicates that this repression involves both DNA methylation-dependent and -independent mechanisms. Transcribed genes with high endogenous levels of H3K4me3, H3K9me3, and H2Bub1, but low H3K9me1, are generally unaffected by SUVR4 activity. Our results imply that SUVR4 is involved in the epigenetic defense mechanism by trimethylating H3K9 to suppress potentially harmful transposon activity.
Basquin, Denis; Spierer, Anne; Begeot, Flora; Koryakov, Dmitry E; Todeschini, Anne-Laure; Ronsseray, Stéphane; Vieira, Cristina; Spierer, Pierre; Delattre, Marion
Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3-7 protein in Drosophila ovaries. We present evidences that Su(var)3-7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3-7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3-7 is required for genome integrity. Females homozygous for Su(var)3-7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3-7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3-7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3-7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3-7 and piwi or aubergine controls important developmental processes independently of transposon silencing.
Begeot, Flora; Koryakov, Dmitry E.; Todeschini, Anne-Laure; Ronsseray, Stéphane; Vieira, Cristina; Spierer, Pierre; Delattre, Marion
Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3–7 protein in Drosophila ovaries. We present evidences that Su(var)3–7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3–7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3–7 is required for genome integrity. Females homozygous for Su(var)3–7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3–7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3–7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3–7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3–7 and piwi or aubergine controls important developmental processes independently of transposon silencing. PMID:24820312
Genome-wide transposon mutagenesis of Proteus mirabilis: Essential genes, fitness factors for catheter-associated urinary tract infection, and the impact of polymicrobial infection on fitness requirements.
Armbruster, Chelsie E; Forsyth-DeOrnellas, Valerie; Johnson, Alexandra O; Smith, Sara N; Zhao, Lili; Wu, Weisheng; Mobley, Harry L T
The Gram-negative bacterium Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Numerous prior studies have uncovered virulence factors for P. mirabilis pathogenicity in a murine model of ascending UTI, but little is known concerning pathogenesis during CAUTI or polymicrobial infection. In this study, we utilized five pools of 10,000 transposon mutants each and transposon insertion-site sequencing (Tn-Seq) to identify the full arsenal of P. mirabilis HI4320 fitness factors for single-species versus polymicrobial CAUTI with Providencia stuartii BE2467. 436 genes in the input pools lacked transposon insertions and were therefore concluded to be essential for P. mirabilis growth in rich medium. 629 genes were identified as P. mirabilis fitness factors during single-species CAUTI. Tn-Seq from coinfection with P. stuartii revealed 217/629 (35%) of the same genes as identified by single-species Tn-Seq, and 1353 additional factors that specifically contribute to colonization during coinfection. Mutants were constructed in eight genes of interest to validate the initial screen: 7/8 (88%) mutants exhibited the expected phenotypes for single-species CAUTI, and 3/3 (100%) validated the expected phenotypes for polymicrobial CAUTI. This approach provided validation of numerous previously described P. mirabilis fitness determinants from an ascending model of UTI, the discovery of novel fitness determinants specifically for CAUTI, and a stringent assessment of how polymicrobial infection influences fitness requirements. For instance, we describe a requirement for branched-chain amino acid biosynthesis by P. mirabilis during coinfection due to high-affinity import of leucine by P. stuartii. Further investigation of genes and pathways that provide a competitive advantage during both single-species and polymicrobial CAUTI will likely provide robust targets for therapeutic intervention to reduce P. mirabilis
Deniger, Drew C; Pasetto, Anna; Tran, Eric; Parkhurst, Maria R; Cohen, Cyrille J; Robbins, Paul F; Cooper, Laurence JN; Rosenberg, Steven A
Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAKS2580F or ERBB2H473Y or the HLA-DQB*0601-restricted neoantigen ERBB2IPE805G were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRβ (mTCRβ) expression. Rapid expansion of mTCRβ+ T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27−CD45RA−) and less-differentiated (CD27+CD45RA+) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens. PMID:26945006
Sveinsson, Saemundur; Gill, Navdeep; Kane, Nolan C; Cronk, Quentin
Transposable elements (TEs) and other repetitive elements are a large and dynamically evolving part of eukaryotic genomes, especially in plants where they can account for a significant proportion of genome size. Their dynamic nature gives them the potential for use in identifying and characterizing crop germplasm. However, their repetitive nature makes them challenging to study using conventional methods of molecular biology. Next generation sequencing and new computational tools have greatly facilitated the investigation of TE variation within species and among closely related species. (i) We generated low-coverage Illumina whole genome shotgun sequencing reads for multiple individuals of cacao (Theobroma cacao) and related species. These reads were analysed using both an alignment/mapping approach and a de novo (graph based clustering) approach. (ii) A standard set of ultra-conserved orthologous sequences (UCOS) standardized TE data between samples and provided phylogenetic information on the relatedness of samples. (iii) The mapping approach proved highly effective within the reference species but underestimated TE abundance in interspecific comparisons relative to the de novo methods. (iv) Individual T. cacao accessions have unique patterns of TE abundance indicating that the TE composition of the genome is evolving actively within this species. (v) LTR/Gypsy elements are the most abundant, comprising c.10% of the genome. (vi) Within T. cacao the retroelement families show an order of magnitude greater sequence variability than the DNA transposon families. (vii) Theobroma grandiflorum has a similar TE composition to T. cacao, but the related genus Herrania is rather different, with LTRs making up a lower proportion of the genome, perhaps because of a massive presence (c. 20%) of distinctive low complexity satellite-like repeats in this genome. (i) Short read alignment/mapping to reference TE contigs provides a simple and effective method of investigating
Laia, Marcelo L; Moreira, Leandro M; Dezajacomo, Juliana; Brigati, Joice B; Ferreira, Cristiano B; Ferro, Maria I T; Silva, Ana C R; Ferro, Jesus A; Oliveira, Julio C F
Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis. Through transposon insertion mutagenesis, 10,000 mutants of Xanthomonas citri subsp. citri strain 306 (Xcc) were obtained, and 3,300 were inoculated in Rangpur lime (Citrus limonia) leaves. Their ability to cause citrus canker was analyzed every 3 days until 21 days after inoculation; a set of 44 mutants showed altered virulence, with 8 presenting a complete loss of causing citrus canker symptoms. Sequencing of the insertion site in all 44 mutants revealed that 35 different ORFs were hit, since some ORFs were hit in more than one mutant, with mutants for the same ORF presenting the same phenotype. An analysis of these ORFs showed that some encoded genes were previously known as related to pathogenicity in phytobacteria and, more interestingly, revealed new genes never implicated with Xanthomonas pathogenicity before, including hypothetical ORFs. Among the 8 mutants with no canker symptoms are the hrpB4 and hrpX genes, two genes that belong to type III secretion system (TTSS), two hypothetical ORFS and, surprisingly, the htrA gene, a gene reported as involved with the virulence process in animal-pathogenic bacteria but not described as involved in phytobacteria virulence. Nucleic acid hybridization using labeled cDNA probes showed that some of the
Wu, Yiming; Hu, Xiaomin; Ge, Yong; Zheng, Dasheng; Yuan, Zhiming
Bacillus sphaericus has been used with great success in mosquito control programs worldwide. Under conditions of nutrient limitation, it undergoes sporulation via a series of well defined morphological stages. However, only a small number of genes involved in sporulation have been identified. To identify genes associated with sporulation, and to understand the relationship between sporulation and crystal protein synthesis, a random mariner-based transposon insertion mutant library of B. sphaericus strain 2297 was constructed and seven sporulation-defective mutants were selected. Sequencing of the DNA flanking of the transposon insertion identified several genes involved in sporulation. The morphologies of mutants were determined by electron microscopy and synthesis of crystal proteins was analyzed by SDS-PAGE and Western blot. Four mutants blocked at early stages of sporulation failed to produce crystal proteins and had lower larvicidal activity. However, the other three mutants were blocked at later stages and were able to form crystal proteins, and the larvicidal activity was similar to wild type. These results indicated that crystal protein synthesis in B. sphaericus is dependent on sporulation initiation. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
Marzo, Mar; Puig, Marta; Ruiz, Alfredo
Galileo is the only transposable element (TE) known to have generated natural chromosomal inversions in the genus Drosophila. It was discovered in Drosophila buzzatii and classified as a Foldback-like element because of its long, internally repetitive, terminal inverted repeats (TIRs) and lack of coding capacity. Here, we characterized a seemingly complete copy of Galileo from the D. buzzatii genome. It is 5,406 bp long, possesses 1,229-bp TIRs, and encodes a 912-aa transposase similar to those of the Drosophila melanogaster 1360 (Hoppel) and P elements. We also searched the recently available genome sequences of 12 Drosophila species for elements similar to Dbuz\\Galileo by using bioinformatic tools. Galileo was found in six species (ananassae, willistoni, peudoobscura, persimilis, virilis, and mojavensis) from the two main lineages within the Drosophila genus. Our observations place Galileo within the P superfamily of cut-and-paste transposons and extend considerably its phylogenetic distribution. The interspecific distribution of Galileo indicates an ancient presence in the genus, but the phylogenetic tree built with the transposase amino acid sequences contrasts significantly with that of the species, indicating lineage sorting and/or horizontal transfer events. Our results also suggest that Foldback-like elements such as Galileo may evolve from DNA-based transposon ancestors by loss of the transposase gene and disproportionate elongation of TIRs.
Ito, Masahiro; Kim, Yun-Gi; Tsuji, Hirokazu; Takahashi, Takuya; Kiwaki, Mayumi; Nomoto, Koji; Danbara, Hirofumi; Okada, Nobuhiko
Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.
Full Text Available Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.
Hirano, Ryoko; Naito, Ken; Fukunaga, Kenji; Watanabe, Kazuo N; Ohsawa, Ryo; Kawase, Makoto
Although the origin and domestication process of foxtail millet (Setaria italica subsp. italica (L.) P. Beauv.) has been studied by several groups, the issue is still ambiguous. It is essential to resolve this issue by studying a large number of accessions with sufficient markers covering the entire genome. Genetic structures were analyzed by transposon display (TD) using 425 accessions of foxtail millet and 12 of the wild ancestor green foxtail (Setaria italica subsp. viridis (L.) P. Beauv.). We used three recently active transposons (TSI-1, TSI-7, and TSI-10) as genome-wide markers and succeeded in demonstrating geographical structures of the foxtail millet. A neighbor-joining dendrogram based on TD grouped the foxtail millet accessions into eight major clusters, each of which consisted of accessions collected from adjacent geographical areas. Eleven out of 12 green foxtail accessions were grouped separately from the clusters of foxtail millet. These results indicated strong regional differentiations and a long history of cultivation in each region. Furthermore, we discuss the relationship between foxtail millet and green foxtail and suggest a monophyletic origin of foxtail millet domestication.
Miki, Takeyoshi; Yamamoto, Yoshihiro; Matsuda, Hideo
We developed a novel, simple, high-throughput method for isolation of genome-wide transposon insertion mutants of Escherichia coli K-12. The basic idea of the method is to randomly disrupt the genes on the DNA fragments cloned on the Kohara library by inserting a mini-transposon first, and then transfer the disrupted genes from the lambda vector to the E. coli chromosome by homologous recombination. Using this method, we constructed a set of 8402 Km(r) cis-diploid mutants harboring a mini-Tn10 insertion mutation and the corresponding wild-type gene on a chromosome, as well as a set of 6954 haploid mutants derived from the cis-diploid mutants. The major advantage of the strategy used is that the indispensable genes or sites for growth can be identified. Preliminary results suggest that 415 open reading frames are indispensable for growth in E. coli cells. A total of 6404 haploid mutants were deposited to Genetic Strains Research Center, National Institute of Genetics, Japan (Chapter 26) and are available for public distribution upon request (http://shigen.lab.nig.ac.jp/ecoli/strain/nbrp/resource.jsp).
Jia, Xianbo; Lin, Xinjian; Chen, Jichen
Current genome walking methods are very time consuming, and many produce non-specific amplification products. To amplify the flanking sequences that are adjacent to Tn5 transposon insertion sites in Serratia marcescens FZSF02, we developed a genome walking method based on TAIL-PCR. This PCR method added a 20-cycle linear amplification step before the exponential amplification step to increase the concentration of the target sequences. Products of the linear amplification and the exponential amplification were diluted 100-fold to decrease the concentration of the templates that cause non-specific amplification. Fast DNA polymerase with a high extension speed was used in this method, and an amplification program was used to rapidly amplify long specific sequences. With this linear and exponential TAIL-PCR (LETAIL-PCR), we successfully obtained products larger than 2 kb from Tn5 transposon insertion mutant strains within 3 h. This method can be widely used in genome walking studies to amplify unknown sequences that are adjacent to known sequences.
Rey, Federico E.; Gonzalez, Mark D.; Cheng, Jiye; Wu, Meng; Ahern, Philip P.; Gordon, Jeffrey I.
Sulfate-reducing bacteria (SRB) colonize the guts of ∼50% of humans. We used genome-wide transposon mutagenesis and insertion-site sequencing, RNA-Seq, plus mass spectrometry to characterize genetic and environmental factors that impact the niche of Desulfovibrio piger, the most common SRB in a surveyed cohort of healthy US adults. Gnotobiotic mice were colonized with an assemblage of sequenced human gut bacterial species with or without D. piger and fed diets with different levels and types ...
Full Text Available Abstract Background Thus far gene therapy strategies for HIV/AIDS have used either conventional retroviral vectors or lentiviral vectors for gene transfer. Although highly efficient, their use poses a certain degree of risk in terms of viral mediated oncogenesis. Sleeping Beauty (SB transposon system offers a non-viral method of gene transfer to avoid this possible risk. With respect to conferring HIV resistance, stable knock down of HIV-1 coreceptors CCR5 and CXCR4 by the use of lentiviral vector delivered siRNAs has proved to be a promising strategy to protect cells from HIV-1 infection. In the current studies our aim is to evaluate the utility of SB system for stable gene transfer of CCR5 and CXCR4 siRNA genes to derive HIV resistant cells as a first step towards using this system for gene therapy. Results Two well characterized siRNAs against the HIV-1 coreceptors CCR5 and CXCR4 were chosen based on their previous efficacy for the SB transposon gene delivery. The siRNA transgenes were incorporated individually into a modified SB transfer plasmid containing a FACS sortable red fluorescence protein (RFP reporter and a drug selectable neomycin resistance gene. Gene transfer was achieved by co-delivery with a construct expressing a hyperactive transposase (HSB5 into the GHOST-R3/X4/R5 cell line, which expresses the major HIV receptor CD4 and and the co-receptors CCR5 and CXCR4. SB constructs expressing CCR5 or CXCR4 siRNAs were also transfected into MAGI-CCR5 or MAGI-CXCR4 cell lines, respectively. Near complete downregulation of CCR5 and CXCR4 surface expression was observed in transfected cells. During viral challenge with X4-tropic (NL4.3 or R5-tropic (BaL HIV-1 strains, the respective transposed cells showed marked viral resistance. Conclusion SB transposon system can be used to deliver siRNA genes for stable gene transfer. The siRNA genes against HIV-1 coreceptors CCR5 and CXCR4 are able to downregulate the respective cell surface proteins
Gorbunova, S.A.; Akhverdyan, V.Z.; Krylov, V.N.
The presence of a large number of clones, which have lost the ability to produce phase, is a feature of PpGl exconjugants, which have acquired markers of the hybrid plasmid containing the genome of the PAO1 phage transposon. Analysis of these clones indicates that they contain plasmids with different defects in the phage genome (point mutations and different deletion lengths which may have an effect on both the phage genome and also plasmid RP4). Mutations of a particular region are selected, the region of early prophage genes. The process of zygotic induction is an essential condition for the arisal of mutants (including deletion mutants). The results of the experiment, which was based on the Luria-Delbruik test, showed that a significant proportion of the mutations, including deletion mutations, arise in the recipient cells PpGl.
Campos, Juliana Coutinho; da Silva, Maria José Félix; dos Santos, Paulo Roberto Nascimento; Barros, Elaine Menezes; Pereira, Mayne de Oliveira; Seco, Bruna Mara Silva; Magagnin, Cibele Massotti; Leiroz, Leonardo Kalab; de Oliveira, Théo Gremen Mimary; de Faria-Júnior, Célio; Cerdeira, Louise Teixeira; Barth, Afonso Luís; Sampaio, Suely Carlos Ferreira; Zavascki, Alexandre Prehn; Poirel, Laurent; Sampaio, Jorge Luiz Mello
In Enterobacteriaceae, the blaNDM genes have been found in many different genetic contexts, and a wide diversity of plasmid scaffolds bearing those genes has been found. In August 2013, we identified NDM-1-producing Escherichia coli and Enterobacter hormaechei strains from a single rectal swab sample from a patient hospitalized in Rio de Janeiro, Brazil, who had no history of travel abroad. Complete DNA sequencing using the Illumina platform and annotation of the two plasmids harboring the blaNDM-1 gene, one from each strain, showed that they belonged to incompatibility groups IncFIIK and IncX3 and harbored a novel transposon named Tn3000. Similar genetic structures have been identified among other isolates in Brazil but also on plasmids from other continents. Our findings suggest that the blaNDM-1 gene may be transmitted by Tn3000 in different parts of the world. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Garcia-Fernàndez, J; Bayascas-Ramírez, J R; Marfany, G; Muñoz-Mármol, A M; Casali, A; Baguñà, J; Saló, E
Several DNA sequences similar to the mariner element were isolated and characterized in the platyhelminthe Dugesia (Girardia) tigrina. They were 1,288 bp long, flanked by two 32 bp-inverted repeats, and contained a single 339 amino acid open-reading frame (ORF) encoding the transposase. The number of copies of this element is approximately 8,000 per haploid genome, constituting a member of the middle-repetitive DNA of Dugesia tigrina. Sequence analysis of several elements showed a high percentage of conservation between the different copies. Most of them presented an intact ORF and the standard signals of actively expressed genes, which suggests that some of them are or have recently been functional transposons. The high degree of similarity shared with other mariner elements from some arthropods, together with the fact that this element is undetectable in other planarian species, strongly suggests a case of horizontal transfer between these two distant phyla.
Full Text Available Recent years have seen major breakthroughs in genome-engineering systems, such as transposon-mediated gene delivery systems and CRISPR-Cas9-mediated genome-editing tools. In these systems, transient expression of auxiliary genes is responsible for permanent genomic modification. For both systems, it would be valuable to select for cells that are likely to undergo stable genome modification. Importantly, in particular for clinical applications of genome-engineered cell products, it will also be of importance to remove those cells that, due to random vector integration, display an unwanted stable expression of the auxiliary gene. Here, we develop a traceless selection system that on the one hand allows efficient enrichment of modified cells, and on the other hand can be used to select against cells that retain expression of the auxiliary gene. The value of this system to produce highly enriched-auxiliary gene-free cell products is demonstrated.
Luo, Wentian; Galvan, Daniel L; Woodard, Lauren E; Dorset, Dan; Levy, Shawn; Wilson, Matthew H
Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.
Okshevsky, Mira; Louw, Matilde Greve; Lamela, Elena Otero; Nilsson, Martin; Tolker-Nielsen, Tim; Meyer, Rikke Louise
Bacillus cereus is one of the most common opportunistic pathogens causing foodborne illness, as well as a common source of contamination in the dairy industry. B. cereus can form robust biofilms on food processing surfaces, resulting in food contamination due to shedding of cells and spores. Despite the medical and industrial relevance of this species, the genetic basis of biofilm formation in B. cereus is not well studied. In order to identify genes required for biofilm formation in this bacterium, we created a library of 5000 + transposon mutants of the biofilm-forming strain B. cereusATCC 10987, using an unbiased mariner transposon approach. The mutant library was screened for the ability to form a pellicle biofilm at the air-media interface, as well as a submerged biofilm at the solid-media interface. A total of 91 genes were identified as essential for biofilm formation. These genes encode functions such as chemotaxis, amino acid metabolism and cellular repair mechanisms, and include numerous genes not previously known to be required for biofilm formation. Although the majority of disrupted genes are not directly responsible for motility, further investigations revealed that the vast majority of the biofilm-deficient mutants were also motility impaired. This observation implicates motility as a pivotal factor in the formation of a biofilm by B. cereus. These results expand our knowledge of the fundamental molecular mechanisms of biofilm formation by B. cereus. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
vonHoldt, Bridgett M; Ji, Sarah S; Aardema, Matthew L; Stahler, Daniel; Udell, Monique A R; Sinsheimer, Janet S
In canines, transposon dynamics have been associated with a hyper-social behavioral syndrome, although the functional mechanism has yet to be described. We investigate the epigenetic and transcriptional consequences of these behavior-associated mobile element insertions in dogs and Yellowstone wolves. We posit that the transposons themselves may not be the causative feature; rather, their transcriptional regulation may exert the functional impact. We survey four outlier transposons associated with hyper-sociability, with the expectation that they are targeted for epigenetic silencing. We predict hyper-methylation of mobile element insertions (MEIs), suggestive that the epigenetic silencing of and not the MEIs themselves may be driving dysregulation of nearby genes. We found that transposon-derived sequences are significantly hyper-methylated, regardless of their copy number or species. Further, we have assessed transcriptome sequence data and found evidence that mobile element insertions impact the expression levels of six genes (WBSCR17, LIMK1, GTF2I, WBSCR27, BAZ1B, and BCL7B), all of which have known roles in human Williams-Beuren syndrome due to changes in copy number, typically hemizygosity. Although further evidence is needed, our results suggest that a few insertions alter local expression at multiple genes, likely through a cis-regulatory mechanism that excludes proximal methylation.
Combination of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 technique with the piggybac transposon system for mouse in utero electroporation to study cortical development.
Cheng, Man; Jin, Xubin; Mu, Lili; Wang, Fangyu; Li, Wei; Zhong, Xiaoling; Liu, Xuan; Shen, Wenchen; Liu, Ying; Zhou, Yan
In utero electroporation (IUE) is commonly used to study cortical development of cerebrum by downregulating or overexpressing genes of interest in neural progenitor cells (NPCs) of small mammals. However, exogenous plasmids are lost or diluted over time. Furthermore, gene knockdown based on short-hairpin RNAs may exert nonspecific effects that lead to aberrant neuronal migration. Genomic engineering by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has great research and therapeutic potentials. Here we integrate the CRISPR/Cas9 components into the piggyBac (PB) transposon system (the CRISPR/Cas9-PB toolkit) for cortical IUEs. The mouse Sry-related HMG box-2 (Sox2) gene was selected as the target for its application. Most transduced cortical NPCs were depleted of SOX2 protein as early as 3 days post-IUE, whereas expressions of SOX1 and PAX6 remained intact. Furthermore, both the WT Cas9 and the D10A nickase mutant Cas9n showed comparable knockout efficiency. Transduced cortical cells were purified with fluorescence-activated cell sorting, and effective gene editing at the Sox2 loci was confirmed. Thus, application of the CRISPR/Cas9-PB toolkit in IUE is a promising strategy to study gene functions in cortical NPCs and their progeny. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Qadri, M I; Flores, C C; Davis, A J; Lichtenstein, C P
The large (14 kb; kb = 10(3) bases) bacterial transposon, Tn7 (encoding resistance to trimethoprim and streptomycin/spectinomycin), has unusual properties. Like other elements, Tn7 transposes with low efficiency and low target-site specificity, but Tn7 also transposes, with high frequency in a unique orientation, to a preferred "attachment" site, called attTn7, in the Escherichia coli chromosome and similarly into plasmids containing attTn7. We developed a novel bacteriophage M13-based assay system to measure the transposition frequency of Tn7 to M13mp phage vectors containing attTn7 on a cloned 1 kb fragment of chromosomal DNA. Phage harvested from a Tn7 donor strain were used to infect recipient bacteria with selection for trimethoprim resistance. Transposition frequency, expressed as the number of trimethoprim-resistant colonies per plaque-forming unit, was found to be approximately 10(-4) to M13mp::attTn7, in contrast to 10(-10) to M13mp recombinants with approximately 1 kb insertions of other, "generic brand", DNA. By deletion analysis of M13mp::attTn7, we show that attTn7 is contained within a 64 base-pair region; sequences adjacent to the actual insertion site and encoding the carboxy terminus of the glmS gene are required. This assay also provided evidence for transposition immunity conferred by the right end of Tn7.
Kashiwa, Takeshi; Suzuki, Tatsuya; Sato, Akira; Akai, Kotaro; Teraoka, Tohru; Komatsu, Ken; Arie, Tsutomu
Emergence of races in Fusarium oxysporum f. sp. lycopersici (Fol) is caused by loss or mutation of at least one avirulence (AVR) gene. The product of AVR1 is a small protein (Avr1) secreted by Fol in tomato xylem sap during infection. This protein triggers Fol race 1 specific resistance (I) in tomato, indicating that AVR1 is an AVR gene. Deletion of AVR1 in race 1 resulted in the emergence of race 2, and an additional mutation in AVR2 generated race 3. Previously, we reported a new biotype of race 3, KoChi-1, in which AVR1 was truncated by a transposon Hormin, which suggested a new route to evolution of races in Fol However, to date no race 2 isolate carrying Hormin-truncated AVR1 has been reported. In this report, we describe such isolates, represented by Chiba-5, in which Hormin insertion occurred in AVR1 at a position different from that in KoChi-1. AVR1 truncation in both isolates resulted in production of defective Avr1 proteins. Chiba-5 and KoChi-1 belong to different phylogenetic clades, A1 and A2, respectively, suggesting that insertion of Hormin in AVR1 in Chiba-5 and KoChi-1 occurred as independent evolutionary events. © FEMS 2016. All rights reserved. For permissions, please e-mail: firstname.lastname@example.org.
Kolacsek, Orsolya; Pergel, Enikő; Varga, Nóra; Apáti, Ágota; Orbán, Tamás I
There are numerous applications of quantitative PCR for both diagnostic and basic research. As in many other techniques the basis of quantification is that comparisons are made between different (unknown and known or reference) specimens of the same entity. When the aim is to compare real quantities of different species in samples, one cannot escape their separate precise absolute quantification. We have established a simple and reliable method for this purpose (Ct shift method) which combines the absolute and the relative approach. It requires a plasmid standard containing both sequences of amplicons to be compared (e.g. the target of interest and the endogenous control). It can serve as a reference sample with equal copies of templates for both targets. Using the ΔΔCt formula we can quantify the exact ratio of the two templates in each unknown sample. The Ct shift method has been successfully applied for transposon gene copy measurements, as well as for comparison of different mRNAs in cDNA samples. This study provides the proof of concept and introduces some potential applications of the method; the absolute nature of results even without the need for real reference samples can contribute to the universality of the method and comparability of different studies. Copyright Â© 2016 Elsevier B.V. All rights reserved.
Salmonella Enteritidis is the world’s leading cause of food borne salmonellosis and illness in people is linked strongly to its contamination of eggs produced by otherwise healthy appearing hens. Salmonella Enteritidis is noted for generating exceptional strain heterogeneity despite having a clonal ...
Full Text Available Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL initiated from a single D. obtusa female. The change in direction and magnitude of Pokey and rRNA gene number did not show a consistent pattern across ∼ 87 generations in the MAL; however, Pokey and rRNA gene number changed in concert. PokeyA and 28S gene number were positively correlated in the isolates from both natural populations and the MAL. PokeyB number was much lower than PokeyA in both MAL and natural population isolates, and showed no correlation with 28S gene number. Preliminary analysis did not detect PokeyB outside rDNA in any isolates and detected only 0 to 4 copies of PokeyA outside rDNA indicating that Pokey may be primarily an rDNA element in D. obtusa. The recombination rate in this species is high and the average size of the rDNA locus is about twice as large as that in other Daphnia species such as D. pulicaria and D. pulex, which may have facilitated expansion of PokeyA to much higher numbers in D. obtusa rDNA than these other species.
Full Text Available Abstract Background Peanut (Arachis hypogaea is an autogamous allotetraploid legume (2n = 4x = 40 that is widely cultivated as a food and oil crop. More than 6,000 DNA markers have been developed in Arachis spp., but high-density linkage maps useful for genetics, genomics, and breeding have not been constructed due to extremely low genetic diversity. Polymorphic marker loci are useful for the construction of such high-density linkage maps. The present study used in silico analysis to develop simple sequence repeat-based and transposon-based markers. Results The use of in silico analysis increased the efficiency of polymorphic marker development by more than 3-fold. In total, 926 (34.2% of 2,702 markers showed polymorphisms between parental lines of the mapping population. Linkage analysis of the 926 markers along with 253 polymorphic markers selected from 4,449 published markers generated 21 linkage groups covering 2,166.4 cM with 1,114 loci. Based on the map thus produced, 23 quantitative trait loci (QTLs for 15 agronomical traits were detected. Another linkage map with 326 loci was also constructed and revealed a relationship between the genotypes of the FAD2 genes and the ratio of oleic/linoleic acid in peanut seed. Conclusions In silico analysis of polymorphisms increased the efficiency of polymorphic marker development, and contributed to the construction of high-density linkage maps in cultivated peanut. The resultant maps were applicable to QTL analysis. Marker subsets and linkage maps developed in this study should be useful for genetics, genomics, and breeding in Arachis. The data are available at the Kazusa DNA Marker Database (http://marker.kazusa.or.jp.
Full Text Available High-throughput sequencing of transposon (Tn libraries created within entire genomes identifies and quantifies the contribution of individual genes and operons to the fitness of organisms in different environments. We used insertion-sequencing (INSeq to analyze the contribution to fitness of all non-essential genes in the chromosome of Pseudomonas aeruginosa strain PA14 based on a library of ∼300,000 individual Tn insertions. In vitro growth in LB provided a baseline for comparison with the survival of the Tn insertion strains following 6 days of colonization of the murine gastrointestinal tract as well as a comparison with Tn-inserts subsequently able to systemically disseminate to the spleen following induction of neutropenia. Sequencing was performed following DNA extraction from the recovered bacteria, digestion with the MmeI restriction enzyme that hydrolyzes DNA 16 bp away from the end of the Tn insert, and fractionation into oligonucleotides of 1,200-1,500 bp that were prepared for high-throughput sequencing. Changes in frequency of Tn inserts into the P. aeruginosa genome were used to quantify in vivo fitness resulting from loss of a gene. 636 genes had <10 sequencing reads in LB, thus defined as unable to grow in this medium. During in vivo infection there were major losses of strains with Tn inserts in almost all known virulence factors, as well as respiration, energy utilization, ion pumps, nutritional genes and prophages. Many new candidates for virulence factors were also identified. There were consistent changes in the recovery of Tn inserts in genes within most operons and Tn insertions into some genes enhanced in vivo fitness. Strikingly, 90% of the non-essential genes were required for in vivo survival following systemic dissemination during neutropenia. These experiments resulted in the identification of the P. aeruginosa strain PA14 genes necessary for optimal survival in the mucosal and systemic environments of a mammalian
Tol2 transposon-mediated transgenesis in the Midas cichlid (Amphilophus citrinellus) - towards understanding gene function and regulatory evolution in an ecological model system for rapid phenotypic diversification.
Kratochwil, Claudius F; Sefton, Maggie M; Liang, Yipeng; Meyer, Axel
The Midas cichlid species complex (Amphilophus spp.) is widely known among evolutionary biologists as a model system for sympatric speciation and adaptive phenotypic divergence within extremely short periods of time (a few hundred generations). The repeated parallel evolution of adaptive phenotypes in this radiation, combined with their near genetic identity, makes them an excellent model for studying phenotypic diversification. While many ecological and evolutionary studies have been performed on Midas cichlids, the molecular basis of specific phenotypes, particularly adaptations, and their underlying coding and cis-regulatory changes have not yet been studied thoroughly. For the first time in any New World cichlid, we use Tol2 transposon-mediated transgenesis in the Midas cichlid (Amphilophus citrinellus). By adapting existing microinjection protocols, we established an effective protocol for transgenesis in Midas cichlids. Embryos were injected with a Tol2 plasmid construct that drives enhanced green fluorescent protein (eGFP) expression under the control of the ubiquitin promoter. The transgene was successfully integrated into the germline, driving strong ubiquitous expression of eGFP in the first transgenic Midas cichlid line. Additionally, we show transient expression of two further transgenic constructs, ubiquitin::tdTomato and mitfa::eGFP. Transgenesis in Midas cichlids will facilitate further investigation of the genetic basis of species-specific traits, many of which are adaptations. Transgenesis is a versatile tool not only for studying regulatory elements such as promoters and enhancers, but also for testing gene function through overexpression of allelic gene variants. As such, it is an important first step in establishing the Midas cichlid as a powerful model for studying adaptive coding and non-coding changes in an ecological and evolutionary context.
Corbo, Joseph C.
On account of the numerous drawbacks of presently existing marker gene systems, we have decided to concentrate on the use of the gusA gene as a marker, since it avoids many of the problems encountered by these other systems. Before I discuss the goals of my work, I would like briefly to describe the beta-gtucuronidase enzyme, its structural gene gusA, and their normal function in E. coli. The gusA gene was originally isolated from E. coli, one of the major constituents of the microflora of the mammalian gastrointestinal tract. In its native host it forms an operon with two other genes, gusB and gusC, and is involved in the uptake and degradation of glucuronidated compounds. Since glucuronidation (the covalent attachment of a glucuronide group) is the principal means of detoxification of xenobiotic compounds in human beings, it is reasonable enough that E. co//should have evolved such an enzyme. By selectively taking up the glucuronated compounds which it encounters in the gut, E. co//acquires a food source, glucuronic acid, which it obtains by hydrolyzing the glucuronide from its aglycone by means of the beta-glucuronidase enzyme. When we assay beta-glucuronidase activity we employ a colorless substrate known as X-gluc which has the chemical formula 5-bromo-4-chloro-3-indolyl beta-D-glucuronide. Upon removal of the glucuronide group the aglycone quickly dimerizes via oxidation to produce an insoluble, deep blue precipitate, which is readily identifiable as the hallmark sign of beta-glucuronidase activity. In this work it was tried to accomplish two related goals: the first goal was to build a marker gene construct using the gusA gene that would meet the requirements for a marker system that I have outlined above; the second goal was to find the best possible marker delivery system that would allow maximal ease of transfer of the marker gene from a host strain to the soil bacterium which is to be marked
Rodić, Nemanja; Burns, Kathleen H
LINE-1 (L1) retrotransposons make up a significant portion of human genomes, with an estimated 500,000 copies per genome. Like other retrotransposons, L1 retrotransposons propagate through RNA sequences that are reverse transcribed into DNA sequences, which are integrated into new genomic loci. L1 somatic insertions have the potential to disrupt the transcriptome by inserting into or nearby genes. By mutating genes and playing a role in epigenetic dysregulation, L1 transposons may contribute to tumorigenesis. Studies of the "mobilome" have lagged behind other tumor characterizations at the sequence, transcript, and epigenetic levels. Here, we consider evidence that L1 retrotransposons may sometimes drive human tumorigenesis.
Full Text Available LINE-1 (L1 retrotransposons make up a significant portion of human genomes, with an estimated 500,000 copies per genome. Like other retrotransposons, L1 retrotransposons propagate through RNA sequences that are reverse transcribed into DNA sequences, which are integrated into new genomic loci. L1 somatic insertions have the potential to disrupt the transcriptome by inserting into or nearby genes. By mutating genes and playing a role in epigenetic dysregulation, L1 transposons may contribute to tumorigenesis. Studies of the "mobilome" have lagged behind other tumor characterizations at the sequence, transcript, and epigenetic levels. Here, we consider evidence that L1 retrotransposons may sometimes drive human tumorigenesis.
Rosconi, Federico; de Vries, Stefan P W; Baig, Abiyad; Fabiano, Elena; Grant, Andrew J
The interior of plants contains microorganisms (referred to as endophytes) that are distinct from those present at the root surface or in the surrounding soil. Herbaspirillum seropedicae strain SmR1, belonging to the betaproteobacteria, is an endophyte that colonizes crops, including rice, maize, sugarcane, and sorghum. Different approaches have revealed genes and pathways regulated during the interactions of H. seropedicae with its plant hosts. However, functional genomic analysis of transposon (Tn) mutants has been hampered by the lack of genetic tools. Here we successfully employed a combination of in vivo high-density mariner Tn mutagenesis and targeted Tn insertion site sequencing (Tn-seq) in H. seropedicae SmR1. The analysis of multiple gene-saturating Tn libraries revealed that 395 genes are essential for the growth of H. seropedicae SmR1 in tryptone-yeast extract medium. A comparative analysis with the Database of Essential Genes (DEG) showed that 25 genes are uniquely essential in H. seropedicae SmR1. The Tn mutagenesis protocol developed and the gene-saturating Tn libraries generated will facilitate elucidation of the genetic mechanisms of the H. seropedicae endophytic lifestyle. A focal point in the study of endophytes is the development of effective biofertilizers that could help to reduce the input of agrochemicals in croplands. Besides the ability to promote plant growth, a good biofertilizer should be successful in colonizing its host and competing against the native microbiota. By using a systematic Tn-based gene-inactivation strategy and massively parallel sequencing of Tn insertion sites (Tn-seq), it is possible to study the fitness of thousands of Tn mutants in a single experiment. We have applied the combination of these techniques to the plant-growth-promoting endophyte Herbaspirillum seropedicae SmR1. The Tn mutant libraries generated will enable studies into the genetic mechanisms of H. seropedicae-plant interactions. The approach that we
The complete genome sequencing of Prevotella intermedia strain OMA14 and a subsequent fine-scale, intra-species genomic comparison reveal an unusual amplification of conjugative and mobile transposons and identify a novel Prevotella-lineage-specific repeat.
Naito, Mariko; Ogura, Yoshitoshi; Itoh, Takehiko; Shoji, Mikio; Okamoto, Masaaki; Hayashi, Tetsuya; Nakayama, Koji
Prevotella intermedia is a pathogenic bacterium involved in periodontal diseases. Here, we present the complete genome sequence of a clinical strain, OMA14, of this bacterium along with the results of comparative genome analysis with strain 17 of the same species whose genome has also been sequenced, but not fully analysed yet. The genomes of both strains consist of two circular chromosomes: the larger chromosomes are similar in size and exhibit a high overall linearity of gene organizations, whereas the smaller chromosomes show a significant size variation and have undergone remarkable genome rearrangements. Unique features of the Pre. intermedia genomes are the presence of a remarkable number of essential genes on the second chromosomes and the abundance of conjugative and mobilizable transposons (CTns and MTns). The CTns/MTns are particularly abundant in the second chromosomes, involved in its extensive genome rearrangement, and have introduced a number of strain-specific genes into each strain. We also found a novel 188-bp repeat sequence that has been highly amplified in Pre. intermedia and are specifically distributed among the Pre. intermedia-related species. These findings expand our understanding of the genetic features of Pre. intermedia and the roles of CTns and MTns in the evolution of bacteria. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.
A Constitutively Mannose-Sensitive Agglutinating Salmonella enterica subsp. enterica Serovar Typhimurium Strain, Carrying a Transposon in the Fimbrial Usher Gene stbC, Exhibits Multidrug Resistance and Flagellated Phenotypes
Full Text Available Static broth culture favors Salmonella enterica subsp. enterica serovar Typhimurium to produce type 1 fimbriae, while solid agar inhibits its expression. A transposon inserted in stbC, which would encode an usher for Stb fimbriae of a non-flagellar Salmonella enterica subsp. enterica serovar Typhimurium LB5010 strain, conferred it to agglutinate yeast cells on both cultures. RT-PCR revealed that the expression of the fimbrial subunit gene fimA, and fimZ, a regulatory gene of fimA, were both increased in the stbC mutant when grown on LB agar; fimW, a repressor gene of fimA, exhibited lower expression. Flagella were observed in the stbC mutant and this phenotype was correlated with the motile phenotype. Microarray data and RT-PCR indicated that the expression of three genes, motA, motB, and cheM, was enhanced in the stbC mutant. The stbC mutant was resistant to several antibiotics, consistent with the finding that expression of yhcQ and ramA was enhanced. A complementation test revealed that transforming a recombinant plasmid possessing the stbC restored the mannose-sensitive agglutination phenotype to the stbC mutant much as that in the parental Salmonella enterica subsp. enterica serovar Typhimurium LB5010 strain, indicating the possibility of an interplay of different fimbrial systems in coordinating their expression.
López, María; Kadlec, Kristina; Schwarz, Stefan; Torres, Carmen
The trimethoprim resistance gene dfrK has been recently described in Staphylococcus aureus, but so far has not been found in other bacteria. A total of 166 enterococci of different species (E. faecium, E. faecalis, E. hirae, E. durans, E. gallinarum, and E. casseliflavus) and origins (food, clinical diseases in humans, healthy humans or animals, and sewage) were studied for their susceptibility to trimethoprim as determined by agar dilution (European Committee on Antimicrobial Susceptibility Testing) and the presence of (a) the dfrK gene and its genetic environment and (b) other dfr genes. The dfrK gene was detected in 49% of the enterococci (64% and 42% of isolates with minimum inhibitory concentrations of ≥2 mg/L or ≤1 mg/L, respectively). The tet(L)-dfrK linkage was detected in 21% of dfrK-positive enterococci. The chromosomal location of the dfrK gene was identified in one E. faecium isolate in which the dfrK was not linked to tet(L) gene but was part of a Tn559 element, which was integrated in the chromosomal radC gene. This Tn559 element was also found in 14 additional isolates. All combinations of dfr genes were detected among the isolates tested (dfrK, dfrG, dfrF, dfrK+dfrG, dfrK+dfrF, dfrF+dfrG, and dfrF+dfrG+dfrK). The gene dfrK gene was found together with other dfr genes in 58% of the tested enterococci. This study suggested an exchange of the trimethoprim resistance gene dfrK between enterococci and staphylococci, as previously observed for the trimethoprim resistance gene dfrG.
Randolph, Lauren N; Bao, Xiaoping; Zhou, Chikai; Lian, Xiaojun
Human pluripotent stem cells (hPSCs) offer tremendous promise in tissue engineering and cell-based therapies due to their unique combination of two properties: pluripotency and unlimited proliferative capacity. However, directed differentiation of hPSCs to clinically relevant cell lineages is needed to achieve the goal of hPSC-based therapies. This requires a deep understanding of how cell signaling pathways converge on the nucleus to control differentiation and the ability to dissect gene function in a temporal manner. Here, we report the use of the PiggyBac transposon and a Tet-On 3G drug-inducible gene expression system to achieve versatile inducible gene expression in hPSC lines. Our new system, XLone, offers improvement over previous Tet-On systems with significantly reduced background expression and increased sensitivity to doxycycline. Transgene expression in hPSCs is tightly regulated in response to doxycycline treatment. In addition, the PiggyBac elements in our XLone construct provide a rapid and efficient strategy for generating stable transgenic hPSCs. Our inducible gene expression PiggyBac transposon system should facilitate the study of gene function and directed differentiation in human stem cells.
Douglas, Darlene S.; Popko, Brian
Forward genetics, the phenotype-driven approach to investigating gene identity and function, has a long history in mouse genetics. Random mutations in the mouse transcend bias about gene function and provide avenues towards unique discoveries. The study of the peripheral nervous system is no exception; from historical strains such as the trembler mouse, which led to the identification of PMP22 as a human disease gene causing multiple forms of peripheral neuropathy, to the more recent identification of the claw paw and sprawling mutations, forward genetics has long been a tool for probing the physiology, pathogenesis, and genetics of the PNS. Even as spontaneous and mutagenized mice continue to enable the identification of novel genes, provide allelic series for detailed functional studies, and generate models useful for clinical research, new methods, such as the piggyBac transposon, are being developed to further harness the power of forward genetics. PMID:18481175
Rice is a major staple food crop and a recognizedmonocotylenedousmodel plant from which gene function discovery is projected to contribute to improvements in a variety of cereals like wheat and maize. The recent release of rough drafts of the rice genome sequence for public
Carda-Diéguez, M; Silva-Hernández, F X; Hubbard, T P; Chao, M C; Waldor, M K; Amaro, C
Vibrio vulnificus can be a highly invasive pathogen capable of spreading from an infection site to the bloodstream, causing sepsis and death. To survive and proliferate in blood, the pathogen requires mechanisms to overcome the innate immune defenses and metabolic limitations of this host niche. We created a high-density transposon mutant library in YJ016, a strain representative of the most virulent V. vulnificus lineage (or phylogroup) and used transposon insertion sequencing (TIS) screens to identify loci that enable the pathogen to survive and proliferate in human serum. Initially, genes underrepresented for insertions were used to estimate the V. vulnificus essential gene set; comparisons of these genes with similar TIS-based classification of underrepresented genes in other vibrios enabled the compilation of a common Vibrio essential gene set. Analysis of the relative abundance of insertion mutants in the library after exposure to serum suggested that genes involved in capsule biogenesis are critical for YJ016 complement resistance. Notably, homologues of two genes required for YJ016 serum-resistance and capsule biogenesis were not previously linked to capsule biogenesis and are largely absent from other V. vulnificus strains. The relative abundance of mutants after exposure to heat inactivated serum was compared with the findings from the serum screen. These comparisons suggest that in both conditions the pathogen relies on its Na + transporting NADH-ubiquinone reductase (NQR) complex and type II secretion system to survive/proliferate within the metabolic constraints of serum. Collectively, our findings reveal the potency of comparative TIS screens to provide knowledge of how a pathogen overcomes the diverse limitations to growth imposed by serum.
Gallego-Bartolomé, Javier; Gardiner, Jason; Liu, Wanlu; Papikian, Ashot; Ghoshal, Basudev; Kuo, Hsuan Yu; Zhao, Jenny Miao-Chi; Jacobsen, Steven E.
DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. Changes in DNA methylation can be heritable and, thus, can lead to the formation of stable epialleles. A well-characterized example of a stable epiallele in plants is fwa, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing up-regulation of FWA and a heritable late-flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA up-regulation, and a heritable late-flowering phenotype. Additional ZF–TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCas9-based targeted demethylation system using the TET1cd and a modified SunTag system. Similar to the ZF–TET1cd fusions, the SunTag–TET1cd system is able to target demethylation and activate gene expression when directed to the FWA or CACTA1 loci. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons. PMID:29444862
Full Text Available We report here the generation of human iPS cell line UKKi009-A from dermal fibroblasts of a patient carrying heterozygous mutation c.3035-3045delTCCCTCGATGC, p.Leu1012Pro (fs*55 in KCNH2 gene leading to long QT syndrome type 2 (LQT2. We used the Sleeping Beauty transposon-based plasmids expressing OSKM along with microRNAs 307/367 to reprogram the fibroblasts. The iPS cells possess pluripotent stem cell characteristics and differentiate to cell lineages of all three germ layers. This cell line can serve as a source for in vitro modeling of LQT2. This cell line is distributed by the European Collection of Authenticated Cell Cultures (ECACC.
Within the literature surrounding nonhuman animals on the one hand and cognitively disabled humans on the other, there is much discussion of where beings that do not satisfy the criteria for personhood fit in our moral deliberations. In the future, we may face a different but related problem: that we might create (or cause the creation of) beings that not only satisfy but exceed these criteria. The question becomes whether these are minimal criteria, or hierarchical, such that those who fulfill them to greater degree should be afforded greater consideration. This article questions the validity and necessity of drawing divisions among beings that satisfy the minimum requirements for personhood; considering how future beings-intelligent androids, synthezoids, even alternate-substrate sentiences-might fit alongside the "baseline" human. I ask whether these alternate beings ought to be considered different to us, and why this may or may not matter in terms of a notion of "human community." The film Blade Runner, concerned in large part with humanity and its key synthezoid antagonist Roy Batty, forms a framing touchstone for my discussion. Batty is stronger, faster, more resilient, and more intelligent than Homo sapiens. His exploits, far beyond the capability of normal humans, are contrasted with his frailty and transient lifespan, his aesthetic appreciation of the sights he has seen, and his burgeoning empathy. Not for nothing does his creator within the mythos term him "more human than human."
Magiorkinis, Gkikas; Belshaw, Robert; Katzourakis, Aris
Almost 8% of the human genome comprises endogenous retroviruses (ERVs). While they have been shown to cause specific pathologies in animals, such as cancer, their association with disease in humans remains controversial. The limited evidence is partly due to the physical and bioethical restrictions surrounding the study of transposons in humans, coupled with the major experimental and bioinformatics challenges surrounding the association of ERVs with disease in general. Two biotechnological landmarks of the past decade provide us with unprecedented research artillery: (i) the ultra-fine sequencing of the human genome and (ii) the emergence of high-throughput sequencing technologies. Here, we critically assemble research about potential pathologies of ERVs in humans. We argue that the time is right to revisit the long-standing questions of human ERV pathogenesis within a robust and carefully structured framework that makes full use of genomic sequence data. We also pose two thought-provoking research questions on potential pathophysiological roles of ERVs with respect to immune escape and regulation. PMID:23938753
Yang, Seong Hwan; Park, Bum; Gang, Yeong Sik; Gal, Won Mo; Baek, Seung Ryeol; Choe, Jeong Hwa; Kim, Dae Sung
This book mentions human engineering, which deals with introduction of human engineering, Man-Machine system like system design, and analysis and evaluation of Man-Machine system, data processing and data input, display, system control of man, human mistake and reliability, human measurement and design of working place, human working, hand tool and manual material handling, condition of working circumstance, working management, working analysis, motion analysis working measurement, and working improvement and design in human engineering.
Full Text Available Piwi-interacting RNAs (piRNAs, a class of 26- to 32-nt non-coding RNAs (ncRNAs, function in germline development, transposon silencing, and epigenetic regulation. We performed deep sequencing and annotation of untreated and periodate-treated small RNA cDNA libraries from human fetal and adult germline and reference somatic tissues. This revealed abundant piRNAs originating from 150 piRNA-encoding genes, including some exhibiting gender-specific expression, in fetal ovary and adult testis—developmental periods coinciding with mitotic cell divisions expanding fetal germ cells prior to meiotic divisions. The absence of reads mapping uniquely to annotated piRNA genes demonstrated their paucity in fetal testis and adult ovary and absence in somatic tissues. We curated human piRNA-expressing regions and defined their precise borders and observed piRNA-guided cleavage of transcripts antisense to some piRNA-producing genes. This study provides insights into sex-specific mammalian piRNA expression and function and serves as a reference for human piRNA analysis and annotation.
There are two classes of transposable elements: DNA transposons and retrotransposons. DNA transposons spread in the genome by “cut and paste” mechanism. In contrast, retrotransposons use copy and paste strategy involving RNA and retrotranscriptase mediated mechanism; these include long interspersed nuclear elements-1 (LINE-1, L1) and short interspersed nuclear elements (SINE). In mammals, in order to maintain genome integrity both types of transposons are tightly repressed. However, some copies of retrotransposons are still active in germ cells contributing to natural variation. Surprisingly, recent reports indicate that also somatic cells support L1 reactivation in early development, in particular in the brain leading to mosaicism. However, whether L1 retrotransposition is a part of other cell lineage developmental programs and its functional significance in the context of cell differentiation remain to be elucidated. To address this question, I investigated whether L1 retrotransposition was occurring during in vitro osteogenic and adipogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). Interestingly, clinical observations have revealed loss of bone density in HIV-infected individuals treated with nucleoside analogs that inhibit HIV retrotranscriptase, as well as the endogenous one encoded by L1s. This observation made us to hypothesize that transposable elements played a positive role in post-natal bone homeostasis. I found that while adipogenesis is “retrotransposition free”, osteogenic differentiation is a “retrotransposition-prone” process and its inhibition blocks its genetic program. Indeed, L1 DNA content does not change during adipogenic differentiation and that of retrotranscriptase does not have any effect on the acquisition of a terminally differentiated phenotype. In contrast, soon after MSCs commitment into pre-osteoblasts, L1 retrotransposable elements increase their expression and actively transpose
Gaay Fortman, B. de
Human rights reflect a determined effort to protect the dignity of each and every human being against abuse of power. This endeavour is as old as human history. What is relatively new is the international venture for the protection of human dignity through internationally accepted legal standards
Concepts and techniques of human reliability have been developed and are used mostly in probabilistic risk assessment. For this, the major application of human reliability assessment has been to identify the human errors which have a significant effect on the overall safety of the system and to quantify the probability of their occurrence. Some of the major issues within human reliability studies are reviewed and it is shown how these are applied to the assessment of human failures in systems. This is done under the following headings; models of human performance used in human reliability assessment, the nature of human error, classification of errors in man-machine systems, practical aspects, human reliability modelling in complex situations, quantification and examination of human reliability, judgement based approaches, holistic techniques and decision analytic approaches. (UK)
Human rights, human development and human security form increasingly important, partly interconnected, partly competitive and misunderstood ethical and policy discourses. Each tries to humanize a pre-existing and unavoidable major discourse of everyday life, policy and politics; each has emerged within the United Nations world; each relies implicitly on a conceptualisation of human need; each has specific strengths. Yet mutual communication, understanding and co-operation are deficient, espec...
The concept of a 'human nature' or 'human natures' retains a central role in theorizing about the human experience. In Homo sapiens it is clear that we have a suite of capacities generated via our evolutionary past, and present, and a flexible capacity to create and sustain particular kinds of cultures and to be shaped by them. Regardless of whether we label these capacities 'human natures' or not, humans occupy a distinctive niche and an evolutionary approach to examining it is critical. At present we are faced with a few different narratives as to exactly what such an evolutionary approach entails. There is a need for a robust and dynamic theoretical toolkit in order to develop a richer, and more nuanced, understanding of the cognitively sophisticated genus Homo and the diverse sorts of niches humans constructed and occupied across the Pleistocene, Holocene, and into the Anthropocene. Here I review current evolutionary approaches to 'human nature', arguing that we benefit from re-framing our investigations via the concept of the human niche and in the context of the extended evolutionary synthesis (EES). While not a replacement of standard evolutionary approaches, this is an expansion and enhancement of our toolkit. I offer brief examples from human evolution in support of these assertions.
Full Text Available Streptococcus suis, a major porcine pathogen, has been receiving growing attention not only for its role in severe and increasingly reported infections in humans, but also for its involvement in drug resistance. Recent studies and the analysis of sequenced genomes have been providing important insights into the S. suis resistome, and have resulted in the identification of resistance determinants for tetracyclines, macrolides, aminoglycosides, chloramphenicol, antifolate drugs, streptothricin, and cadmium salts. Resistance gene-carrying genetic elements described so far include integrative and conjugative elements, transposons, genomic islands, phages, and chimeric elements. Some of these elements are similar to those reported in major streptococcal pathogens such as Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus agalactiae and share the same chromosomal insertion sites. The available information strongly suggests that S. suis is an important antibiotic resistance reservoir that can contribute to the spread of resistance genes to the above-mentioned streptococci. S. suis is thus a paradigmatic example of possible intersections between animal and human resistomes.
Siegel - Rozenblit, Dina; Zaitch, Damian
Human smuggling is based on a consensus between smuggler, smuggled, and his/her family (which usually guarantees or effectuates payment). However, unauthorized immigrants are violating immigration laws and human smugglers are profiting from enabling illegal immigration. Both human smuggling and its
Hora, S.; Neill, R.; Williams, R.; Bauser, M.; Channell, J.
This paper focused on the possible approaches to evaluating the impacts of human intrusion on nuclear waste disposal. Several major issues were reviewed. First, it was noted that human intrusion could be addressed either quantitatively through performance assessments or qualitatively through design requirements. Second, it was decided that it was impossible to construct a complete set of possible future human intrusion scenarios. Third, the question of when the effect of possible human intrusion should be considered, before or after site selection was reviewed. Finally, the time frame over which human intrusion should be considered was discussed
Human Technology and Human Affects This year Samsung introduced a mobile phone with "Soul". It was made with a human touch and included itself a magical touch. Which function does technology and affects get in everyday aesthetics like this, its images and interactions included this presentation...... will ask and try to answer. The mobile phone and its devices are depicted as being able to make a unique human presence, interaction, and affect. The medium, the technology is a necessary helper to get towards this very special and lost humanity. Without the technology, no special humanity - soul....... The paper will investigate how technology, humanity, affects, and synaesthesia are presented and combined with examples from everyday aesthetics, e.g. early computer tv-commercial, net-commercial for mobile phones. Technology and affects point, is the conclusion, towards a forgotten pre-human and not he...
Söderlund-Venermo, Maria; Young, Neal S.
SUMMARY Parvovirus B19 (B19V) and human bocavirus 1 (HBoV1), members of the large Parvoviridae family, are human pathogens responsible for a variety of diseases. For B19V in particular, host features determine disease manifestations. These viruses are prevalent worldwide and are culturable in vitro, and serological and molecular assays are available but require careful interpretation of results. Additional human parvoviruses, including HBoV2 to -4, human parvovirus 4 (PARV4), and human bufavirus (BuV) are also reviewed. The full spectrum of parvovirus disease in humans has yet to be established. Candidate recombinant B19V vaccines have been developed but may not be commercially feasible. We review relevant features of the molecular and cellular biology of these viruses, and the human immune response that they elicit, which have allowed a deep understanding of pathophysiology. PMID:27806994
Riccardo De Santis
Full Text Available Human pluripotent stem cells (PSCs are widely used for in vitro disease modeling. One of the challenges in the field is represented by the ability of converting human PSCs into specific disease-relevant cell types. The nervous system is composed of a wide variety of neuronal types with selective vulnerability in neurodegenerative diseases. This is particularly relevant for motor neuron diseases, in which different motor neurons populations show a different susceptibility to degeneration. Here we developed a fast and efficient method to convert human induced Pluripotent Stem Cells into cranial motor neurons of the branchiomotor and visceral motor subtype. These populations represent the motor neuron subgroup that is primarily affected by a severe form of amyotrophic lateral sclerosis with bulbar onset and worst prognosis. This goal was achieved by stable integration of an inducible vector, based on the piggyBac transposon, allowing controlled activation of Ngn2, Isl1 and Phox2a (NIP. The NIP module effectively produced electrophysiologically active cranial motor neurons. Our method can be easily extended to PSCs carrying disease-associated mutations, thus providing a useful tool to shed light on the cellular and molecular bases of selective motor neuron vulnerability in pathological conditions. Keywords: Spinal motor neuron, Cranial motor neuron, Induced pluripotent stem cells, Amyotrophic lateral sclerosis, Phox2a, piggyBac
The faculty of Holy Names High School developed an interdisciplinary human rights program with school-wide activities focusing on three selected themes: the United Nations Universal Declaration of Human Rights, in conjunction with Human Rights Week; Food; and Women. This article outlines major program activities. (SJL)
Peña Avalos, B.L. de la
Most human tumors have mutations in genes of the RAS small GTPase protein family. RAS works as a molecular switch for signaling pathways that modulate many aspects of cell behavior, including proliferation, differentiation, motility and death. Oncogenic mutations in RAS prevent GTP hydrolysis, locking RAS in a permanently active state, being the most common mutations in HRAS, KRAS and NRAS. The human RAS family consists of at least 36 different genes, many of which have been scarcely studied. One of these relatively unknown genes is ERAS (ES cell-expressed RAS), which is a constitutively active RAS protein, localized in chromosome X and expressed only in embryonic cells, being undetectable in adult tissues. New high throughput technologies have made it possible to screen complete cancer genomes for identification of mutations associated to cancer. Using the Sleeping Beauty (SB) transposon system, ERAS was identified as a putative novel oncogene in non-melanoma skin and breast cancers. The major aim of this project is to determine the general characteristics of ERAS as a putative novel human oncogene in skin and breast cells. Forced expression of ERAS results in drastic changes in cell shape, proliferation and motility. When ERAS is overexpressed in skin and breast human cells it is mainly localized in the cytoplasmic membrane. ERAS activates the phosphatidylinositol-3-OH kinase (PI3K) pathway but not the mitogen-activated protein kinase (MAPK) pathway. ERAS-expressing cells suffer spontaneous morphologic and phenotypic EMT-like changes, including cytoskeleton reorganization, vimentin and N-cadherin up-regulation and down-regulation of E-cadherin, which can be associated with increased malignancy, and invasive and metastatic potential. Our results suggest that inappropriate expression of ERAS lead to transformation of human cells. (Author)
D.R. Gasper (Des)
markdownabstractAbstract: Human rights, human development and human security form increasingly important, partly interconnected, partly competitive and misunderstood ethical and policy discourses. Each tries to humanize a pre-existing and unavoidable major discourse of everyday life, policy and
Llamas, Bastien; Willerslev, Eske; Orlando, Ludovic Antoine Alexandre
The field of human ancient DNA (aDNA) has moved from mitochondrial sequencing that suffered from contamination and provided limited biological insights, to become a fully genomic discipline that is changing our conception of human history. Recent successes include the sequencing of extinct homini...
Nielsen, Charlotte Marie Bisgaard
years' campaigns suggests that the theory of communication underlying the campaign has its basis in mechanical action rather than in human communication. The practice of 'Communication design' is investigated in relation to this metaphorical 'machine thinking' model of communication and contrasted...... with the human-centered theory of communication advocated by integrationism....
Grosen, Anders; Nielsen, Peder Harbjerg
finansiel og human kapital. Den traditionelle rådgivnings snævre synsvinkel kan føre til forkerte investeringsråd. Der skal derfor opfordres til, at de finansielle virksomheder i tilrettelæggelsen af deres rådgivning af private kunder systematisk inddrager den humane kapitals størrelse og karakteristika i...
Betson, Martha; Søe, Martin Jensen; Nejsum, Peter
Human trichuriasis is a neglected tropical disease which affects hundreds of millions of people worldwide and is particularly prevalent among children living in areas where sanitation is poor. This review examines the current knowledge on the taxonomy, genetics and phylogeography of human Trichuris...
Jordan R Plews
Full Text Available Several methods have been used to induce somatic cells to re-enter the pluripotent state. Viral transduction of reprogramming genes yields higher efficiency but involves random insertions of viral sequences into the human genome. Although induced pluripotent stem (iPS cells can be obtained with the removable PiggyBac transposon system or an episomal system, both approaches still use DNA constructs so that resulting cell lines need to be thoroughly analyzed to confirm they are free of harmful genetic modification. Thus a method to change cell fate without using DNA will be very useful in regenerative medicine.In this study, we synthesized mRNAs encoding OCT4, SOX2, cMYC, KLF4 and SV40 large T (LT and electroporated them into human fibroblast cells. Upon transfection, fibroblasts expressed these factors at levels comparable to, or higher than those in human embryonic stem (ES cells. Ectopically expressed OCT4 localized to the cell nucleus within 4 hours after mRNA introduction. Transfecting fibroblasts with a mixture of mRNAs encoding all five factors significantly increased the expression of endogenous OCT4, NANOG, DNMT3β, REX1 and SALL4. When such transfected fibroblasts were also exposed to several small molecules (valproic acid, BIX01294 and 5'-aza-2'-deoxycytidine and cultured in human embryonic stem cell (ES medium they formed small aggregates positive for alkaline phosphatase activity and OCT4 protein within 30 days.Our results demonstrate that mRNA transfection can be a useful approach to precisely control the protein expression level and short-term expression of reprogramming factors is sufficient to activate pluripotency genes in differentiated cells.
Full Text Available Alginates pertain to organic polysaccharides that have been extensively used in food- and medicine-related industries. The present study obtained alginates from an alginate overproducing Pseudomonas aeruginosa PAO1 mutant by screening transposon mutagenesis libraries. The interaction between bacterial and seaweed alginates and gut microbiota were further studied by using an in vitro batch fermentation system. Thin-layer chromatography (TLC analysis indicated that both bacterial and seaweed alginates can be completely degraded by fecal bacteria isolated from study volunteers, indicating that a minor structural difference between bacterial and seaweed alginates (O-acetylation and lack of G-G blocks didn't affect the digestion of alginates by human microbiota. Although, the digestion of bacterial and seaweed alginates was attributed to different Bacteroides xylanisolvens strains, they harbored similar alginate lyase genes. Genus Bacteroides with alginate-degrading capability were enriched in growth medium containing bacterial or seaweed alginates after in vitro fermentation. Short-chain fatty acid (SCFA production in both bacterial and seaweed alginates was also comparable, but was significantly higher than the same medium using starch. In summary, the present study has isolated an alginate-overproducing P. aeruginosa mutant strain. Both seaweed and bacterial alginates were degraded by human gut microbiota, and their regulatory function on gut microbiota was similar.
Bai, Shaofeng; Chen, Huahai; Zhu, Liying; Liu, Wei; Yu, Hongwei D; Wang, Xin; Yin, Yeshi
Alginates pertain to organic polysaccharides that have been extensively used in food- and medicine-related industries. The present study obtained alginates from an alginate overproducing Pseudomonas aeruginosa PAO1 mutant by screening transposon mutagenesis libraries. The interaction between bacterial and seaweed alginates and gut microbiota were further studied by using an in vitro batch fermentation system. Thin-layer chromatography (TLC) analysis indicated that both bacterial and seaweed alginates can be completely degraded by fecal bacteria isolated from study volunteers, indicating that a minor structural difference between bacterial and seaweed alginates (O-acetylation and lack of G-G blocks) didn't affect the digestion of alginates by human microbiota. Although, the digestion of bacterial and seaweed alginates was attributed to different Bacteroides xylanisolvens strains, they harbored similar alginate lyase genes. Genus Bacteroides with alginate-degrading capability were enriched in growth medium containing bacterial or seaweed alginates after in vitro fermentation. Short-chain fatty acid (SCFA) production in both bacterial and seaweed alginates was also comparable, but was significantly higher than the same medium using starch. In summary, the present study has isolated an alginate-overproducing P. aeruginosa mutant strain. Both seaweed and bacterial alginates were degraded by human gut microbiota, and their regulatory function on gut microbiota was similar.
, and preserving material to study, as an object of study in its own right, as an analytical tool, or for collaborating, and for disseminating results. The term "digital humanities" was coined around 2001, and gained currency within academia in the following years. However, computers had been used within......Digital humanities is an umbrella term for theories, methodologies, and practices related to humanities scholarship that use the digital computer as an integrated and essential part of its research and teaching activities. The computer can be used for establishing, finding, collecting...
What if people could play computer games and accomplish work without even realizing it? What if billions of people collaborated to solve important problems for humanity or generate training data for computers? My work aims at a general paradigm for doing exactly that: utilizing human processing power to solve computational problems in a distributed manner. In particular, I focus on harnessing human time and energy for addressing problems that computers cannot yet solve. Although computers have advanced dramatically in many respects over the last 50 years, they still do not possess the basic conceptual intelligence or perceptual capabilities...
Thomas Nagel in `The Absurd' (Nagel 1971) mentions the future expunction of the human species as a `metaphor' for our ability to see our lives from the outside, which he claims is one source of our sense of life's absurdity. I argue that the future expunction (not to be confused with extinction) of everything human - indeed of everything biological in a terran sense - is not a mere metaphor but a physical certainty under the laws of nature. The causal processes by which human expunction will take place are presented in some empirical detail, so that philosophers cannot dismiss it as merely speculative. I also argue that appeals to anthropic principles or to forms of mystical cosmology are of no plausible avail in the face of human expunction under the laws of physics.
Johnson, Judith A; Williams, Erin D
.... Scientists in other labs, including Harvard University and the University of California at San Francisco, intend to produce cloned human embryos in order to derive stem cells for medical research...
Franco, María Pía; Mulder, Maximilian; Gilman, Robert H.; Smits, Henk L.
Human brucellosis still presents scientists and clinicians with several challenges, such as the understanding of pathogenic mechanisms of Brucella spp, the identification of markers for disease severity, progression, and treatment response, and the development of improved treatment regimens.
Van Niekerk, Cornelia W
Full Text Available risk of deaths and injuries by drowning in floods and migration- related health effects. • Increased migration, which can result in human suffering, human rights violations, conflicts and political instability. • Loss of property and livelihoods.... The vulnerability of settlements in southern Africa is impacted by various and complex socio-economic processes related to the cultural, political and institutional contexts and demographic pressure, as well as specific high-risk zones susceptible to flash floods...
Human Fertilization and Embryology Authority (HFEA). A team of scientists headed by Alison Murdoch at the University of Newcastle received permission...not yet reported success in isolating stem cells from a cloned human embryo. A research team headed by Ian Wilmut at the University of Edinburgh...research group, headed by Douglas Melton and Kevin Eggan, submitted their proposal to a Harvard committee composed of ethicists, scientists and public
The study of human cognition encompasses the study of all mental phenomena, from the receipt and interpretation of sensory information to the final control of the motor system in the performance of action. The cognitive scientist examines all intermediary processes, including thought, decision making, and memory and including the effects of motivation, states of arousal and stress, the study of language, and the effects of social factors. The field therefore ranges over an enormous territory, covering all that is known or that should be known about human behavior. It is not possible to summarize the current state of knowledge about cognition with any great confidence that we know the correct answer about any aspect of the work. Nontheless, models provide good characterizations of certain aspects of the data and situations. Even if these models should prove to be incorrect, they do provide good approximate descriptions of people's behavior in some situations, and these approximations will still apply even when the underlying theories have changed. A quick description is provided of models within a number of areas of human cognition and skill and some general theoretical frameworks with which to view human cognition. The frameworks are qualitative descriptions that provide a way to view the development of more detailed, quantitative models and, most important, a way of thinking about human performance and skill
In the first part of this paper a short history of Western humanisms (Socrates, Pico della Mirandola, Descartes, Kant) is presented. As far as these humanisms rest on a fixation of the ‘humanum’ they are metaphysical, although they might radically differ from each other. The second part deals with the present debate on trans- and posthumanism in the context of some breath-taking developments in science and technology.Angeletics, a theory of messengers and messages, intends to give an answer t...
While absorbing visible light energy for photosynthesis, plants are unavoidably exposed to ultraviolet radiation, which is particularly harmful at shorter wavelengths (UV-B radiation). Ozone depletion in the atmosphere means that plants receive episodic or steadily increasing doses of UV-B, which damages their photosynthetic reaction centres, crosslinks cellular proteins, and induces mutagenic DNA lesions. Plant adaptive mechanisms of shielding and repair are therefore critical to survival — for example, somatic tissues of maize and Arabidopsis defective in phenolic sunscreen pigments incur increased DNA damage, and mutants defective in DNA repair are killed by UV-B
Dupeyron, Mathilde; Leclercq, Sébastien; Cerveau, Nicolas; Bouchon, Didier; Gilbert, Clément
Horizontal transfer of transposable elements (HTT) is increasingly appreciated as an important source of genome and species evolution in eukaryotes. However, our understanding of HTT dynamics is still poor in eukaryotes because the diversity of species for which whole genome sequences are available is biased and does not reflect the global eukaryote diversity. In this study we characterized two Mariner transposable elements (TEs) in the genome of several terrestrial crustacean isopods, a group of animals particularly underrepresented in genome databases. The two elements have a patchy distribution in the arthropod tree and they are highly similar (>93% over the entire length of the element) to insect TEs (Diptera and Hymenoptera), some of which were previously described in Ceratitis rosa (Crmar2) and Drosophila biarmipes (Mariner-5_Dbi). In addition, phylogenetic analyses and comparisons of TE versus orthologous gene distances at various phylogenetic levels revealed that the taxonomic distribution of the two elements is incompatible with vertical inheritance. We conclude that the two Mariner TEs each underwent at least three HTT events. Both elements were transferred once between isopod crustaceans and insects and at least once between isopod crustacean species. Crmar2 was also transferred between tephritid and drosophilid flies and Mariner-5 underwent HT between hymenopterans and dipterans. We demonstrate that these various HTTs took place recently (most likely within the last 3 million years), and propose iridoviruses and/or Wolbachia endosymbionts as potential vectors of these transfers.
Fischer, Thea Kølsen; Harvala, Heli; Midgley, Sofie
Infections with human parechoviruses (HPeV) are highly prevalent, particularly in neonates, where they may cause substantial morbidity and mortality. The clinical presentation of HPeV infection is often indistinguishable from that of enterovirus (EV) infection and may vary from mild disease...
and self-reflective democracy. Contemporary humanities have adopted a new orientation towards practices, and it is not clear how this fits with the ideals of ‘Bildung’ and ‘pure science’. A possible theoretical framework for this orientation towards practices could be found in John Dewey’s pragmatic...
Amin, Md Nurul; Kroeze, Carolien; Strokal, Maryna
Many people practice open defecation in south Asia. As a result, lot of human waste containing nutrients such as nitrogen (N) and phosphorus (P) enter rivers. Rivers transport these nutrients to coastal waters, resulting in marine pollution. This source of nutrient pollution is, however, ignored in
Wilson, David McKay
The shadowy, criminal nature of human trafficking makes evaluating its nature and scope difficult. The U.S. State Department and anti-trafficking groups estimate that worldwide some 27 million people are caught in a form of forced servitude today. Public awareness of modern-day slavery is gaining momentum thanks to new abolitionist efforts. Among…
Nielsen, Charlotte Marie Bisgaard
years' campaigns suggests that the theory of communication underlying the campaign has its basis in mechanical action rather than in human communication. The practice of 'Communication design' is investigated in relation to this metaphorical 'machine thinking' model of communication and contrasted...
Wharram, C. C.
In this essay C. C. Wharram argues that Terence's concept of translation as a form of "contamination" anticipates recent developments in philosophy, ecology, and translation studies. Placing these divergent fields of inquiry into dialogue enables us read Terence's well-known statement "I am a human being--I deem nothing…
Full Text Available This paper firstly explores some theories of Human Rights justification and then assents to the theory that Human Rights is based on justified moral values. In order to justify moral values, Aristotle’s approach called “Function Argument” is reviewed. Propounding this argument, the writer attempts to show that all analysis of human identity will directly contribute to the man’s view of his rights. Not only Human rights is really determined by human function or human distinguishing characteristic i.e. human identity, but in the world of knowledge the proper method to know human rights is to know human being himself. n cloning violates man’s rights due to two reasons: damage of human identity and violation of the right to be unique. Attempting to clarify the nature of human cloning, this article examines the aspects to be claimed to violate human rights and evaluates the strength of the reasons for this claim. این مقاله پس از بررسی اجمالی برخی از نظریههای توجیه حقوق بشر، نظریة ابتنای آن بر ارزشهای اخلاقی موجّه را میپذیرد. دربارة چگونگی توجیه ارزش اخلاقی، رویکرد ارسطو که به «برهان ارگن» موسوم است، مورد بحث و بررسی قرار میگیرد. مؤلف با طرح این برهان میکوشد نشان دهد ارائه هرگونه تحلیل از هویت انسان در نگرش آدمی به حقوق خود تأثیر مستقیم خواهد گذاشت. حقوق آدمی نه فقط از ناحیة کارویژه یا فصل ممیز وی (هویت انسان تعیّن واقعی میگیرد، بلکه در عالم معرفت هم راه درست شناخت حقوق بشر، شناخت خود انسان است.
D.R. Gasper (Des)
textabstractHuman rights, human development and human security form increasingly important, partly interconnected, partly competitive and misunderstood ethical and policy discourses. Each tries to humanize a pre-existing and unavoidable major discourse of everyday life, policy and politics; each
Human face as a physical human recognition can be used as a unique identity for computer to recognize human by transforming human face with face algorithm as simple text number which can be primary key for human. Human face as single identity for human will be done by making a huge and large world centre human face database, where the human face around the world will be recorded from time to time and from generation to generation. Architecture database will be divided become human face image ...
Human rights are rapidly entering the academic curriculum, with programs appearing all over the country--including at Duke, Harvard, Northeastern, and Stanford Universities; the Massachusetts Institute of Technology; the Universities of Chicago, of Connecticut, of California at Berkeley, and of Minnesota; and Trinity College. Most of these…
This book resulted from the activity of Task Force 4.2 - 'Human Reliability'. This group was established on February 27th, 1986, at the plenary meeting of the Technical Reliability Committee of VDI, within the framework of the joint committee of VDI on industrial systems technology - GIS. It is composed of representatives of industry, representatives of research institutes, of technical control boards and universities, whose job it is to study how man fits into the technical side of the world of work and to optimize this interaction. In a total of 17 sessions, information from the part of ergonomy dealing with human reliability in using technical systems at work was exchanged, and different methods for its evaluation were examined and analyzed. The outcome of this work was systematized and compiled in this book. (orig.) [de
The book presents an integrative review of paleoneurology, the study of endocranial morphology in fossil species. The main focus is on showing how computed methods can be used to support advances in evolutionary neuroanatomy, paleoanthropology and archaeology and how they have contributed to creating a completely new perspective in cognitive neuroscience. Moreover, thanks to its multidisciplinary approach, the book addresses students and researchers approaching human paleoneurology from different angles and for different purposes, such as biologists, physicians, anthropologists, archaeologists
Human life is a staggeringly strange thing. On the surface of a ball of rock falling around a nuclear fireball in the blackness of a vacuum the laws of nature conspired to create a naked ape that can look up at the stars and wonder where it came from. What is a human being? Objectively, nothing of consequence. Particles of dust in an infinite arena, present for an instant in eternity. Clumps of atoms in a universe with more galaxies than people. And yet a human being is necessary for the question itself to exist, and the presence of a question in the universe - any question - is the most wonderful thing. Questions require minds, and minds bring meaning. What is meaning? I don't know, except that the universe and every pointless speck inside it means something to me. I am astonished by the existence of a single atom, and find my civilisation to be an outrageous imprint on reality. I don't understand it. Nobody does, but it makes me smile. This book asks questions about our origins, our destiny, and our place i...
Rey, Federico E; Gonzalez, Mark D; Cheng, Jiye; Wu, Meng; Ahern, Philip P; Gordon, Jeffrey I
Sulfate-reducing bacteria (SRB) colonize the guts of ∼50% of humans. We used genome-wide transposon mutagenesis and insertion-site sequencing, RNA-Seq, plus mass spectrometry to characterize genetic and environmental factors that impact the niche of Desulfovibrio piger, the most common SRB in a surveyed cohort of healthy US adults. Gnotobiotic mice were colonized with an assemblage of sequenced human gut bacterial species with or without D. piger and fed diets with different levels and types of carbohydrates and sulfur sources. Diet was a major determinant of functions expressed by this artificial nine-member community and of the genes that impact D. piger fitness; the latter includes high- and low-affinity systems for using ammonia, a limiting resource for D. piger in mice consuming a polysaccharide-rich diet. Although genes involved in hydrogen consumption and sulfate reduction are necessary for its colonization, varying dietary-free sulfate levels did not significantly alter levels of D. piger, which can obtain sulfate from the host in part via cross-feeding mediated by Bacteroides-encoded sulfatases. Chondroitin sulfate, a common dietary supplement, increased D. piger and H2S levels without compromising gut barrier integrity. A chondroitin sulfate-supplemented diet together with D. piger impacted the assemblage's substrate utilization preferences, allowing consumption of more reduced carbon sources and increasing the abundance of the H2-producing Actinobacterium, Collinsella aerofaciens. Our findings provide genetic and metabolic details of how this H2-consuming SRB shapes the responses of a microbiota to diet ingredients and a framework for examining how individuals lacking D. piger differ from those who harbor it.
Sanchez-Larrayoz, Amaro F; Elhosseiny, Noha M; Chevrette, Marc G; Fu, Yang; Giunta, Peter; Spallanzani, Raúl G; Ravi, Keerthikka; Pier, Gerald B; Lory, Stephen; Maira-Litrán, Tomás
Acinetobacter baumannii is a bacterial pathogen with increasing impact in healthcare settings, due in part to this organism's resistance to many antimicrobial agents, with pneumonia and bacteremia as the most common manifestations of disease. A significant proportion of clinically relevant A. baumannii strains are resistant to killing by normal human serum (NHS), an observation supported in this study by showing that 12 out of 15 genetically diverse strains of A. baumannii are resistant to NHS killing. To expand our understanding of the genetic basis of A. baumannii serum resistance, a transposon (Tn) sequencing (Tn-seq) approach was used to identify genes contributing to this trait. An ordered Tn library in strain AB5075 with insertions in every nonessential gene was subjected to selection in NHS. We identified 50 genes essential for the survival of A. baumannii in NHS, including already known serum resistance factors, and many novel genes not previously associated with serum resistance. This latter group included the maintenance of lipid asymmetry genetic pathway as a key determinant in protecting A. baumannii from the bactericidal activity of NHS via the alternative complement pathway. Follow-up studies validated the role of eight additional genes identified by Tn-seq in A. baumannii resistance to killing by NHS but not by normal mouse serum, highlighting the human species specificity of A. baumannii serum resistance. The identification of a large number of genes essential for serum resistance in A. baumannii indicates the degree of complexity needed for this phenotype, which might reflect a general pattern that pathogens rely on to cause serious infections. Copyright © 2017 by The American Association of Immunologists, Inc.
Aktaş, Tuğçe; Avşar Ilık, İbrahim; Maticzka, Daniel; Bhardwaj, Vivek; Pessoa Rodrigues, Cecilia; Mittler, Gerhard; Manke, Thomas; Backofen, Rolf; Akhtar, Asifa
Transposable elements are viewed as 'selfish genetic elements', yet they contribute to gene regulation and genome evolution in diverse ways. More than half of the human genome consists of transposable elements. Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome. Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability. Alu elements are the main targets of the RNA-editing enzyme ADAR and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC, but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant nuclear RNA helicase, binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post
Vőfély, Gergő; Berecz, Tünde; Szabó, Eszter; Szebényi, Kornélia; Hathy, Edit; Orbán, Tamás I; Sarkadi, Balázs; Homolya, László; Marchetto, Maria C; Réthelyi, János M; Apáti, Ágota
Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation. Copyright © 2018 Elsevier Inc. All rights reserved.
Marchetto, Maria C N; Narvaiza, Iñigo; Denli, Ahmet M; Benner, Christopher; Lazzarini, Thomas A; Nathanson, Jason L; Paquola, Apuã C M; Desai, Keval N; Herai, Roberto H; Weitzman, Matthew D; Yeo, Gene W; Muotri, Alysson R; Gage, Fred H
Identifying cellular and molecular differences between human and non-human primates (NHPs) is essential to the basic understanding of the evolution and diversity of our own species. Until now, preserved tissues have been the main source for most comparative studies between humans, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). However, these tissue samples do not fairly represent the distinctive traits of live cell behaviour and are not amenable to genetic manipulation. We propose that induced pluripotent stem (iPS) cells could be a unique biological resource to determine relevant phenotypical differences between human and NHPs, and that those differences could have potential adaptation and speciation value. Here we describe the generation and initial characterization of iPS cells from chimpanzees and bonobos as new tools to explore factors that may have contributed to great ape evolution. Comparative gene expression analysis of human and NHP iPS cells revealed differences in the regulation of long interspersed element-1 (L1, also known as LINE-1) transposons. A force of change in mammalian evolution, L1 elements are retrotransposons that have remained active during primate evolution. Decreased levels of L1-restricting factors APOBEC3B (also known as A3B) and PIWIL2 (ref. 7) in NHP iPS cells correlated with increased L1 mobility and endogenous L1 messenger RNA levels. Moreover, results from the manipulation of A3B and PIWIL2 levels in iPS cells supported a causal inverse relationship between levels of these proteins and L1 retrotransposition. Finally, we found increased copy numbers of species-specific L1 elements in the genome of chimpanzees compared to humans, supporting the idea that increased L1 mobility in NHPs is not limited to iPS cells in culture and may have also occurred in the germ line or embryonic cells developmentally upstream to germline specification during primate evolution. We propose that differences in L1 mobility may have
Full Text Available NANO: New American Notes Online: An Interdisciplinary Academic Journal for Big Ideas in a Small World. This special issue shows how both public and digital humanities research can be rendered more persuasive through engagement with cultures beyond the academy. More specifically, the aim of this special issue is to demonstrate how investments in technologies and computation are not necessarily antithetical to investments in critical theory and social justice.
Le Grange, L.
In this article I initiate a debate into the (de)merits of human capital theory and human capability theory and discuss implications of the debate for higher education. Human capital theory holds that economic growth depends on investment in education and that economic growth is the basis for improving the quality of human life. Human capable…
Toft, Tanya Søndergaard
The article proposes the urban digital gallery as an opportunity to explore the relationship between ‘human’ and ‘technology,’ through the programming of media architecture. It takes a curatorial perspective when proposing an ontological shift from considering media facades as visual spectacles...... agency and a sense of being by way of dematerializing architecture. This is achieved by way of programming the symbolic to provide new emotional realizations and situations of enlightenment in the public audience. This reflects a greater potential to humanize the digital in media architecture....
Ito, Ryoji; Takahashi, Takeshi; Ito, Mamoru
Humanized mice are superior to rodents for preclinical evaluation of the efficacy and safety of drug candidates using human cells or tissues. During the past decade, humanized mouse technology has been greatly advanced by the establishment of novel platforms of genetically modified immunodeficient mice. Several human diseases can be recapitulated using humanized mice due to the improved engraftment and differentiation capacity of human cells or tissues. In this review, we discuss current advanced humanized mouse models that recapitulate human diseases including cancer, allergy, and graft-versus-host disease. © 2017 Wiley Periodicals, Inc.
Andersen, Claus Y; Ezcurra, Diego
In the menstrual cycle, the mid-cycle surge of gonadotropins (both luteinising hormone [LH] and follicle-stimulating hormone [FSH]) signals the initiation of the periovulatory interval, during which the follicle augments progesterone production and begins to luteinise, ultimately leading to the r......In the menstrual cycle, the mid-cycle surge of gonadotropins (both luteinising hormone [LH] and follicle-stimulating hormone [FSH]) signals the initiation of the periovulatory interval, during which the follicle augments progesterone production and begins to luteinise, ultimately leading...... reviews current knowledge of the regulation of progesterone in the human ovary during the follicular phase and highlights areas where knowledge remains limited. In this review, we provide in-depth information outlining the regulation and function of gonadotropins in the complicated area of steroidogenesis...
Handley, Holly A. H.; Houston, Nancy P.
The NATO Human View is a system architectural viewpoint that focuses on the human as part of a system. Its purpose is to capture the human requirements and to inform on how the human impacts the system design. The viewpoint contains seven static models that include different aspects of the human element, such as roles, tasks, constraints, training and metrics. It also includes a Human Dynamics component to perform simulations of the human system under design. One of the static models, termed Human Networks, focuses on the human-to-human communication patterns that occur as a result of ad hoc or deliberate team formation, especially teams distributed across space and time. Parameters of human teams that effect system performance can be captured in this model. Human centered aspects of networks, such as differences in operational tempo (sense of urgency), priorities (common goal), and team history (knowledge of the other team members), can be incorporated. The information captured in the Human Network static model can then be included in the Human Dynamics component so that the impact of distributed teams is represented in the simulation. As the NATO militaries transform to a more networked force, the Human View architecture is an important tool that can be used to make recommendations on the proper mix of technological innovations and human interactions.
This article argues that the digital humanities can be seen as a humanities project in a time of significant change in the academy. The background is a number of scholarly, educational and technical challenges, the multiple epistemic traditions linked to the digital humanities, the potential reach of the field across and outside the humanities,…
Full Text Available The physical and social realities, mental biases and limitations of being human differentiate human brands from others. It is their very humanness that introduces risk while generating the ability for enhanced returns. Four particular human characteristics can create imbalance or inconsistency between the person and the brand: mortality, hubris, unpredictability and social embeddedness. None of these qualities manifest in traditional non-human brands, and all of them present risks requiring active managerial attention. Rather than treating humans as brands and making humans into brands for sale in the commercial marketplace, our framework forces a focus on keeping a balance between the person and the personified object.
Full Text Available Catholic Church and most of Muslims believe that human cloning is in contrast with human rights. They argue that applying Somatic Nuclear Transfer Technique or so-called cloning to humans is against human dignity. Their main reason is that the cloned person would be a copy or shadow of another person and lack his or her identity and uniqueness. They also argue that in the process of cloning human beings would be treated as laboratory mice. This article tries to evaluate this kind of argumentation and shows that the "human dignity" expression in the relevant writings is vague and has been used inappropriately. مسیحیان و برخی از مسلمانان استدلال میکنند که کاربست تکنیک شبیهسازی ناقض کرامت انسانی است. این دلیل خود به صورتهای مختلفی بیان میشود، مانند آنکه انسان موضوع آزمایشهای علمی قرار میگیرد و با او مانند حیوانات رفتار میشود. گاه نیز تغییر نحوة تولید مثل، مایة نقض کرامت انسانی قلمداد میگردد و گاه به مسئلة از بین رفتن هویت فردی اشاره میشود. نگارنده در دو قسمت، دیدگاه مسیحیان و مسلمانان را در این باره نقل و تحلیل کرده است و کوشیده است نشان دهد که استناد به مفهوم کرامت انسانی در این جا مبهم و ناگویاست و مخالفان کوشش دقیقی در جهت تبیین دلیل خود به عمل نیاوردهاند.
Nielsen, Hans Jørn
overgangen fra trykkekultur til digital kultur. For det første problemstillingen omkring digitalisering af litterær kulturarv med fokus på kodning og tagging af teksten samt organisering i hypertekststrukturer. For det andet reorganiseringen af det digitale dokument i dataelementer og database. For det......Artiklen præsenterer først nogle generelle problemstillinger omkring Digital Humanities (DH) med det formål at undersøge dem nærmere i relation til konkrete eksempler på forskellige digitaliseringsmåder og ændringer i dokumentproduktion. I en nærmere afgrænsning vælger artiklen den tendens i DH......, der betragter DH som forbundet med "making" og "building" af digitale objekter og former. Dette kan også karakteriseres som DH som praktisk-produktiv vending. Artiklen har valgt tre typer af digitalisering. De er valgt ud fra, at de skal repræsentere forskellige måder at håndtere digitaliseringen på...
Hibberd, Matthew C. [Washington Univ. School of Medicine, St. Louis, MO (United States). Center for Genome Sciences and Systems Biology, Center for Gut Microbiome and Nutrition Research; Wu, Meng [Washington Univ. School of Medicine, St. Louis, MO (United States). Center for Genome Sciences and Systems Biology; Rodionov, Dmitry A. [Russian Academy of Sciences (RAS), Moscow (Russian Federation). A.A. Kharkevich Inst. for Information Transmission Problems; Sanford Burnham Prebys Medical Discovery Inst., La Jolla, CA (United States); Li, Xiaoqing [Sanford Burnham Prebys Medical Discovery Inst., La Jolla, CA (United States); Cheng, Jiye [Washington Univ. School of Medicine, St. Louis, MO (United States). Center for Genome Sciences and Systems Biology, Center for Gut Microbiome and Nutrition Researc; Griffin, Nicholas W. [Washington Univ. School of Medicine, St. Louis, MO (United States). Center for Genome Sciences and Systems Biology, Center for Gut Microbiome and Nutrition Researc; Barratt, Michael J. [Washington Univ. School of Medicine, St. Louis, MO (United States). Center for Genome Sciences and Systems Biology, Center for Gut Microbiome and Nutrition Researc; Giannone, Richard J. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Chemical Sciences Division; Hettich, Robert L. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Chemical Sciences Division; Osterman, Andrei L. [Sanford Burnham Prebys Medical Discovery Inst., La Jolla, CA (United States); Gordon, Jeffrey I. [Washington Univ. School of Medicine, St. Louis, MO (United States). Center for Genome Sciences and Systems Biology, Center for Gut Microbiome and Nutrition Researc
Micronutrient deficiencies afflict two billion people. And while the impact of these imbalances on host biology has been studied extensively, much less is known about their effects on the developing or adult gut microbiota. Thus, we established a community of 44 cultured, sequenced human gut-derived bacterial species in gnotobiotic mice and fed the animals a defined, micronutrient-sufficient diet, followed by a derivative diet devoid of vitamin A, folate, iron or zinc, followed by return to the sufficient diet. Acute vitamin A deficiency had the largest effect on community structure and meta-transcriptome, with Bacteroides vulgatus, a prominent responder, increasing its abundance in the absence of vitamin A, and manifesting transcriptional changes involving various metabolic pathways. Applying retinol selection to a library of 30,300 B. vulgatus transposon mutants revealed that disruption of acrR abrogated retinol sensitivity. Genetic complementation studies, microbial RNA-Seq, and transcription factor binding assays disclosed that AcrR functions as a repressor of an adjacent AcrAB-TolC efflux system plus other members of its regulon. Retinol efflux measurements in wild-type, acrR-mutant, and complemented acrR mutant strains, plus treatment with a pharmacologic inhibitor of the efflux system, revealed that AcrAB-TolC is a determinant of retinol and bile acid sensitivity. We associated acute vitamin A deficiency with altered bile acid metabolism in vivo, raising the possibility that retinol, bile acid metabolites, and AcrAB-TolC interact to influence the fitness of B. vulgatus and perhaps other microbiota members. This type of preclinical model can help develop mechanistic insights about and more effective treatment strategies for micronutrient deficiencies.
Full Text Available DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the "gold standard" of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.
Wang, Gang; Yang, Luhan; Grishin, Dennis; Rios, Xavier; Ye, Lillian Y; Hu, Yong; Li, Kai; Zhang, Donghui; Church, George M; Pu, William T
Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA), donor DNA and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%.
Jeong, Byeong Yong; Lee Dong Kyeong
These are the titles of each chapter. They are as in the following; design of human-centerdness, human machine system, information processing process, sense of human, user interface, elements of human body, vital dynamics, measurement of reaction of human body, estimation and management of working environment, mental characteristic of human, human error, group, organization and leadership, safety supervision, process analysis, time studying, work sampling, work factor and methods time measurement, introduction of muscular skeletal disease and program of preventive management.
Jeong, Byeong Yong; Lee Dong Kyeong
These are the titles of each chapter. They are as in the following; design of human-centerdness, human machine system, information processing process, sense of human, user interface, elements of human body, vital dynamics, measurement of reaction of human body, estimation and management of working environment, mental characteristic of human, human error, group, organization and leadership, safety supervision, process analysis, time studying, work sampling, work factor and methods time measurement, introduction of muscular skeletal disease and program of preventive management.
N. S. Shilovskaya
Full Text Available Article is devoted to the distinction of humanism and anthropocentrism which is based on the parity of the person and being. Genetic communication of humanism and anthropocentrism and their historical break comes to light.
Recent studies of the human mind debunk the myth that humans only use 10-20 percent of the human mind. A healthy human mind uses up to 90 percent...way. They will eat what is in front of them to satiate their appetite not knowing if there is anymore food for the future. Humans can predict
Gaay Fortman, B. de
The success or failure of processes of democratization cannot be detached from processes of development related to the aspirations of people at the grassroots. Human rights, in a more theoretical terminology, require human development in order to enhance human security.
Dutton, J.W.; Brown, W.R.
The Human Factors concept is a focused effort directed at those activities which require human involvement. Training is, by its nature, an activity totally dependent on the Human Factor. This paper identifies several concerns significant to training situations and discusses how Human Factor awareness can increase the quality of learning. Psychology in the training arena is applied Human Factors. Training is a method of communication represented by sender, medium, and receiver. Two-thirds of this communications model involves the human element directly
Forsythe, J Chris [Sandia Park, NM; Xavier, Patrick G [Albuquerque, NM; Abbott, Robert G [Albuquerque, NM; Brannon, Nathan G [Albuquerque, NM; Bernard, Michael L [Tijeras, NM; Speed, Ann E [Albuquerque, NM
Digital technology utilizing a cognitive model based on human naturalistic decision-making processes, including pattern recognition and episodic memory, can reduce the dependency of human-machine interactions on the abilities of a human user and can enable a machine to more closely emulate human-like responses. Such a cognitive model can enable digital technology to use cognitive capacities fundamental to human-like communication and cooperation to interact with humans.
Amoh, Yasuyuki; Kanoh, Maho; Niiyama, Shiro; Hamada, Yuko; Kawahara, Katsumasa; Sato, Yuichi; Hoffman, Robert M; Katsuoka, Kensei
The optimal source of stem cells for regenerative medicine is a major question. Embryonic stem (ES) cells have shown promise for pluripotency but have ethical issues and potential to form teratomas. Pluripotent stem cells have been produced from skin cells by either viral-, plasmid- or transposon-mediated gene transfer. These stem cells have been termed induced pluripotent stem cells or iPS cells. iPS cells may also have malignant potential and are inefficiently produced. Embryonic stem cells may not be suited for individualized therapy, since they can undergo immunologic rejection. To address these fundamental problems, our group is developing hair follicle pluripotent stem (hfPS) cells. Our previous studies have shown that mouse hfPS cells can differentiate to neurons, glial cells in vitro, and other cell types, and can promote nerve and spinal cord regeneration in vivo. hfPS cells are located above the hair follicle bulge in what we have termed the hfPS cell area (hfPSA) and are nestin positive and keratin 15 (K-15) negative. Human hfPS cells can also differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In the present study, human hfPS cells were transplanted in the severed sciatic nerve of the mouse where they differentiated into glial fibrillary-acidic-protein (GFAP)-positive Schwann cells and promoted the recovery of pre-existing axons, leading to nerve generation. The regenerated nerve recovered function and, upon electrical stimulation, contracted the gastrocnemius muscle. The hfPS cells can be readily isolated from the human scalp, thereby providing an accessible, autologous and safe source of stem cells for regenerative medicine that have important advantages over ES or iPS cells. (c) 2009 Wiley-Liss, Inc.
Full Text Available What does it mean to be human in the 21st century? The pull of engineering on every aspect of our lives, the impact of machines on how we represent ourselves, the influence of computers on our understanding of free-will, individuality and species, and the effect of microorganisms on our behaviour are so great that one cannot discourse on humanity and humanities without considering their entanglement with technology and with the multiple new dimensions of reality that it opens up. The future of humanities should take into account AI, bacteria, software, viruses (both organic and inorganic, hardware, machine language, parasites, big data, monitors, pixels, swarms systems and the Internet. One cannot think of humanity and humanities as distinct from technology anymore.
Frydenlund, Knut; And Others
Eleven articles examine human rights in Europe. Topics include unemployment, human rights legislation, role of the Council of Europe in promoting human rights, labor unions, migrant workers, human dignity in industralized societies, and international violence. Journal available from Council of Europe, Directorate of Press and Information, 67006…
The human factor's reliability program was at Slovenske elektrarne, a.s. (SE) nuclear power plants. introduced as one of the components Initiatives of Excellent Performance in 2011. The initiative's goal was to increase the reliability of both people and facilities, in response to 3 major areas of improvement - Need for improvement of the results, Troubleshooting support, Supporting the achievement of the company's goals. The human agent's reliability program is in practice included: - Tools to prevent human error; - Managerial observation and coaching; - Human factor analysis; -Quick information about the event with a human agent; -Human reliability timeline and performance indicators; - Basic, periodic and extraordinary training in human factor reliability(authors)
Wheaton, Elizabeth M; Schauer, Edward J; Galli, Thomas V
Because freedom of choice and economic gain are at the heart of productivity, human trafficking impedes national and international economic growth. Within the next 10 years, crime experts expect human trafficking to surpass drug and arms trafficking in its incidence, cost to human well-being, and profitability to criminals (Schauer and Wheaton, 2006: 164-165). The loss of agency from human trafficking as well as from modern slavery is the result of human vulnerability (Bales, 2000: 15). As people become vulnerable to exploitation and businesses continually seek the lowest-cost labour sources, trafficking human beings generates profit and a market for human trafficking is created. This paper presents an economic model of human trafficking that encompasses all known economic factors that affect human trafficking both across and within national borders. We envision human trafficking as a monopolistically competitive industry in which traffickers act as intermediaries between vulnerable individuals and employers by supplying differentiated products to employers. In the human trafficking market, the consumers are employers of trafficked labour and the products are human beings. Using a rational-choice framework of human trafficking we explain the social situations that shape relocation and working decisions of vulnerable populations leading to human trafficking, the impetus for being a trafficker, and the decisions by employers of trafficked individuals. The goal of this paper is to provide a common ground upon which policymakers and researchers can collaborate to decrease the incidence of trafficking in humans.
Literature and medicine is a discipline within medical humanities, which challenges medicine to reconfigure its scientific model to become interdisciplinary, and be disciplined by arts and humanities as well as science. The psychological, emotional, spiritual and physical are inextricably linked in people, inevitably entailing provisionality,…
Vahdat, Mehrnoosh; Oneto, L.; Ghio, A; Anguita, D.; Funk, M.; Rauterberg, G.W.M.
In Machine Learning (ML), the learning process of an algo- rithm given a set of evidences is studied via complexity measures. The way towards using ML complexity measures in the Human Learning (HL) domain has been paved by a previous study, which introduced Human Rademacher Complexity (HRC): in this
Human errors have a major contribution to the risks for industrial accidents. Accidents have provided important lesson making it possible to build safer systems. In avoiding human errors it is necessary to adapt the systems to their operators. The complexity of modern industrial systems is however increasing the danger of system accidents. Models of the human operator have been proposed, but the models are not able to give accurate predictions of human performance. Human errors can never be eliminated, but their frequency can be decreased by systematic efforts. The paper gives a brief summary of research in human error and it concludes with suggestions for further work. (orig.)
Nathan P McNulty
Full Text Available The human gut microbiota is an important metabolic organ, yet little is known about how its individual species interact, establish dominant positions, and respond to changes in environmental factors such as diet. In this study, gnotobiotic mice were colonized with an artificial microbiota comprising 12 sequenced human gut bacterial species and fed oscillating diets of disparate composition. Rapid, reproducible, and reversible changes in the structure of this assemblage were observed. Time-series microbial RNA-Seq analyses revealed staggered functional responses to diet shifts throughout the assemblage that were heavily focused on carbohydrate and amino acid metabolism. High-resolution shotgun metaproteomics confirmed many of these responses at a protein level. One member, Bacteroides cellulosilyticus WH2, proved exceptionally fit regardless of diet. Its genome encoded more carbohydrate active enzymes than any previously sequenced member of the Bacteroidetes. Transcriptional profiling indicated that B. cellulosilyticus WH2 is an adaptive forager that tailors its versatile carbohydrate utilization strategy to available dietary polysaccharides, with a strong emphasis on plant-derived xylans abundant in dietary staples like cereal grains. Two highly expressed, diet-specific polysaccharide utilization loci (PULs in B. cellulosilyticus WH2 were identified, one with characteristics of xylan utilization systems. Introduction of a B. cellulosilyticus WH2 library comprising >90,000 isogenic transposon mutants into gnotobiotic mice, along with the other artificial community members, confirmed that these loci represent critical diet-specific fitness determinants. Carbohydrates that trigger dramatic increases in expression of these two loci and many of the organism's 111 other predicted PULs were identified by RNA-Seq during in vitro growth on 31 distinct carbohydrate substrates, allowing us to better interpret in vivo RNA-Seq and proteomics data. These
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This book assesses the scientific value and merit of research on human genetic differences--including a collection of DNA samples that represents the whole of human genetic diversity--and the ethical...
The ROE is divided into 5 themes: Air, Water, Land, Human Exposure and Health and Ecological Condition. From these themes, the report indicators address fundamental questions that the ROE attempts to answer. For human health there are 3 questions.
IOANA - JULIETA JOSAN
Full Text Available The purpose of this paper is to analyze human resources in terms of quantitative and qualitative side with special focus on the human capital accumulation influence. The paper examines the human resources trough human capital accumulation in terms of modern theory of human resources, educational capital, health, unemployment and migration. The findings presented in this work are based on theoretical economy publications and data collected from research materials. Sources of information include: documents from organizations - the EUROSTAT, INSSE - studies from publications, books, periodicals, and the Internet. The paper describes and analyzes human resources characteristics, human resource capacities, social and economic benefits of human capital accumulation based on economy, and the government plans and policies on health, education and labor market.
Human bites present a high risk of infection. Besides the bacteria which can cause infection, there is ... the wound extends below the skin. Anytime a human bite has broken the skin, seek medical attention.
... Consumers Consumer Information by Audience For Women HPV (human papillomavirus) Share Tweet Linkedin Pin it More sharing ... Español In Chamorro In Urdu In Vietnamese HPV (human papillomavirus) is a sexually transmitted virus. It is ...
Why get vaccinated?HPV vaccine prevents infection with human papillomavirus (HPV) types that are associated with cause ... at http://www.cdc.gov/hpv. HPV Vaccine (Human Papillomavirus) Information Statement. U.S. Department of Health and ...
U.S. Environmental Protection Agency — Human land uses may have major impacts on ecosystems, affecting biodiversity, habitat, air and water quality. The human use index (also known as U-index) is the...
U.S. Environmental Protection Agency — Human land uses may have major impacts on ecosystems, affecting biodiversity, habitat, air and water quality. The human use index (also known as U-index) is the...
Harden, Mallory E; Munger, Karl
Human papillomaviruses are small DNA viruses with a tropism for squamous epithelia. A unique aspect of human papillomavirus molecular biology involves dependence on the differentiation status of the host epithelial cell to complete the viral lifecycle. A small group of these viruses are the etiologic agents of several types of human cancers, including oral and anogenital tract carcinomas. This review focuses on the basic molecular biology of human papillomaviruses. Copyright © 2016 Elsevier B.V. All rights reserved.
Dannenberg, Roger B.
Human Computer Music Performance (HCMP) is the study of music performance by live human performers and real-time computer-based performers. One goal of HCMP is to create a highly autonomous artificial performer that can fill the role of a human, especially in a popular music setting. This will require advances in automated music listening and understanding, new representations for music, techniques for music synchronization, real-time human-computer communication, music generation, sound synt...
Humanities Review Journal is published in June and December by Humanities Research Forum. The Journal publishes original, well-researched papers, review essays, interviews, resume, and commentaries, which offer new insights into the various disciplines in the Humanities. The focus is on issues about Africa.
Svendsen, Mette N.; Gjødsbøl, Iben M.; Dam, Mie S.
At the heart of anthropology and the social sciences lies a notion of human existence according to which humans and animals share the basic need for food, but only humans have the capacity for morality. Based on fieldwork in a pig laboratory, a neonatal intensive care unit (NICU), and a dementia ...
de Vries, Jeroen; Abelmann, Leon; Manz, A; Elwenspoek, Michael Curt
“The Human Document Project‿ is a project which tries to answer all of the questions related to preserving information about the human race for tens of generations of humans to come or maybe even for a future intelligence which can emerge in the coming thousands of years. This document mainly
Parker, Donald G.; Capella, Barry John
In March 1984, the first issue of "Esprit," a semi-annual humanities magazine for the 56 two-year colleges in New York State, was published. The magazine seeks to confront the apparent decline of student interest in the humanities, community doubts about the relevance of the humanities, and the seeming indifference to the special truths…
Presents a human rights glossary that includes definitions of basic terms, treaties, charters, and groups/organizations that have been featured in previous articles in this edition of "Update on Law-Related Education"; the human rights terms have been compiled as part of the celebration of the Universal Declaration of Human Rights…
Alan R. Templeton
Full Text Available It has been argued that human evolution has stopped because humans now adapt to their environment via cultural evolution and not biological evolution. However, all organisms adapt to their environment, and humans are no exception. Culture defines much of the human environment, so cultural evolution has actually led to adaptive evolution in humans. Examples are given to illustrate the rapid pace of adaptive evolution in response to cultural innovations. These adaptive responses have important implications for infectious diseases, Mendelian genetic diseases, and systemic diseases in current human populations. Moreover, evolution proceeds by mechanisms other than natural selection. The recent growth in human population size has greatly increased the reservoir of mutational variants in the human gene pool, thereby enhancing the potential for human evolution. The increase in human population size coupled with our increased capacity to move across the globe has induced a rapid and ongoing evolutionary shift in how genetic variation is distributed within and among local human populations. In particular, genetic differences between human populations are rapidly diminishing and individual heterozygosity is increasing, with beneficial health effects. Finally, even when cultural evolution eliminates selection on a trait, the trait can still evolve due to natural selection on other traits. Our traits are not isolated, independent units, but rather are integrated into a functional whole, so selection on one trait can cause evolution to occur on another trait, sometimes with mildly maladaptive consequences.
Walsh, Kenneth R.; Hoque, Md Tamjidul; Williams, Kim H.
Human Machine Learning Symbiosis is a cooperative system where both the human learner and the machine learner learn from each other to create an effective and efficient learning environment adapted to the needs of the human learner. Such a system can be used in online learning modules so that the modules adapt to each learner's learning state both…
Rösing, Lilian Munk
The article puts forward an aesthetic and psychoanalytic analysis of Titian's painting, The Flaying of Marsyas, arguing that the painting is a reflection on the human subject as a being constituted by skin and by a core of non-humanity. The analysis is partly an answer to Melanie Hart's (2007) ar...... of the 'Muselmann', and Anton Ehrenzweig's psychoanalytic theory of artistic creation. Whereas Hart is focusing on form and colour, I also turn my attention towards the texture of the painting....
This book debates the universe, the development of new technologies in the 21st century and the future of the human race. Dr Bolonkin shows that a human soul is only the information in a person's head. He offers a new unique method for re-writing the main brain information in chips without any damage to the human brain. This is the scientific prediction of the non-biological (electronic) civilization and immortality of the human being. Such a prognosis is predicated upon a new law, discovered by the author, for the development of complex systems. According to this law, every self-copying system tends to be more complex than the previous system, provided that all external conditions remain the same. The consequences are disastrous: humanity will be replaced by a new civilization created by intellectual robots (which Dr Bolonkin refers to as "E-humans" and "E-beings"). These creatures, whose intellectual and mechanical abilities will far exceed those of man, will require neither food nor oxygen to sustain their...
Full Text Available The currently available human tumor xenograft models permit modeling of human cancers in vivo, but in immunocompromised hosts. Here we report a humanized mouse (hu-mouse model made by transplantation of human fetal thymic tissue plus hematopoietic stem cells transduced with a leukemia-associated fusion gene MLL-AF9. In addition to normal human lymphohematopoietic reconstitution as seen in non-leukemic hu-mice, these hu-mice showed spontaneous development of B-cell acute lymphoblastic leukemia (B-ALL, which was transplantable to secondary recipients with an autologous human immune system. Using this model, we show that lymphopenia markedly improves the antitumor efficacy of recipient leukocyte infusion (RLI, a GVHD-free immunotherapy that induces antitumor responses in association with rejection of donor chimerism in mixed allogeneic chimeras. Our data demonstrate the potential of this leukemic hu-mouse model in modeling leukemia immunotherapy, and suggest that RLI may offer a safe treatment option for leukemia patients with severe lymphopenia.
Chiapperino, Luca; Boniolo, Giovanni
This paper questions different conceptions of Medical Humanities in order to provide a clearer understanding of what they are and why they matter. Building upon former attempts, we defend a conception of Medical Humanities as a humanistic problem-based approach to medicine aiming at influencing its nature and practice. In particular, we discuss three main conceptual issues regarding the overall nature of this discipline: (i) a problem-driven approach to Medical Humanities; (ii) the need for an integration of Medical Humanities into medicine; (iii) the methodological requirements that could render Medical Humanities an effective framework for medical decision-making.
Lazarovici, M; Trentzsch, H; Prückner, S
The concept of human factors is commonly used in the context of patient safety and medical errors, all too often ambiguously. In actual fact, the term comprises a wide range of meanings from human-machine interfaces through human performance and limitations up to the point of working process design; however, human factors prevail as a substantial cause of error in complex systems. This article presents the full range of the term human factors from the (emergency) medical perspective. Based on the so-called Swiss cheese model by Reason, we explain the different types of error, what promotes their emergence and on which level of the model error prevention can be initiated.
Glynn, Pierre D.
Integrated Environmental Modelling (IEM) is an invaluable tool for understanding the complex, dynamic ecosystems that house our natural resources and control our environments. Human behaviour affects the ways in which the science of IEM is assembled and used for meaningful societal applications. In particular, human biases and heuristics reflect adaptation and experiential learning to issues with frequent, sharply distinguished, feedbacks. Unfortunately, human behaviour is not adapted to the more diffusely experienced problems that IEM typically seeks to address. Twelve biases are identified that affect IEM (and science in general). These biases are supported by personal observations and by the findings of behavioural scientists. A process for critical analysis is proposed that addresses some human challenges of IEM and solicits explicit description of (1) represented processes and information, (2) unrepresented processes and information, and (3) accounting for, and cognizance of, potential human biases. Several other suggestions are also made that generally complement maintaining attitudes of watchful humility, open-mindedness, honesty and transparent accountability. These suggestions include (1) creating a new area of study in the behavioural biogeosciences, (2) using structured processes for engaging the modelling and stakeholder communities in IEM, and (3) using ‘red teams’ to increase resilience of IEM constructs and use.
Luciana Helena Antoniassi da Silva
Full Text Available The human respiratory syncytial virus (hRSV and the human metapneumovírus (hMPV are main etiological agents of acute respiratory infections (ARI. The ARI is an important cause of childhood morbidity and mortality worldwide. hRSV and hMPV are members of the Paramyxoviridae. They are enveloped, non-segmented viruses, with negative-sense single stranded genomes. Respiratory syncytial virus (hRSV is the best characterized agent viral of this group, associated with respiratory diseases in lower respiratory tract. Recently, a new human pathogen belonging to the subfamily Pneumovirinae was identified, the human metapneumovirus (hMPV, which is structurally similar to the hRSV, in genomic organization, viral structure, antigenicity and clinical symptoms. The subfamily Pneumovirinae contains two genera: genus Pneumovirus contains hRSV, the bovine (bRSV, as well as the ovine and caprine respiratory syncytial virus and pneumonia virus of mice, the second genus Metapneumovirus, consists of avian metapneumovirus (aMPV and human metapneumovirus (hMPV. In this work, we present a brief narrative review of the literature on important aspects of the biology, epidemiology and clinical manifestations of infections by two respiratory viruses.
Karsai, Márton; Kaski, Kimmo
This book provides a comprehensive overview on emergent bursty patterns in the dynamics of human behaviour. It presents common and alternative understanding of the investigated phenomena, and points out open questions worthy of further investigations. The book is structured as follows. In the introduction the authors discuss the motivation of the field, describe bursty phenomena in case of human behaviour, and relate it to other disciplines. The second chapter addresses the measures commonly used to characterise heterogeneous signals, bursty human dynamics, temporal paths, and correlated behaviour. These definitions are first introduced to set the basis for the discussion of the third chapter about the observations of bursty human patterns in the dynamics of individuals, dyadic interactions, and collective behaviour. The subsequent fourth chapter discusses the models of bursty human dynamics. Various mechanisms have been proposed about the source of the heterogeneities in human dynamics, which leads to the in...
Regev, Aviv; Teichmann, Sarah A; Lander, Eric S; Amit, Ido; Benoist, Christophe; Birney, Ewan; Bodenmiller, Bernd; Campbell, Peter; Carninci, Piero; Clatworthy, Menna; Clevers, Hans; Deplancke, Bart; Dunham, Ian; Eberwine, James; Eils, Roland; Enard, Wolfgang; Farmer, Andrew; Fugger, Lars; Göttgens, Berthold; Hacohen, Nir; Haniffa, Muzlifah; Hemberg, Martin; Kim, Seung; Klenerman, Paul; Kriegstein, Arnold; Lein, Ed; Linnarsson, Sten; Lundberg, Emma; Lundeberg, Joakim; Majumder, Partha; Marioni, John C; Merad, Miriam; Mhlanga, Musa; Nawijn, Martijn; Netea, Mihai; Nolan, Garry; Pe'er, Dana; Phillipakis, Anthony; Ponting, Chris P; Quake, Stephen; Reik, Wolf; Rozenblatt-Rosen, Orit; Sanes, Joshua; Satija, Rahul; Schumacher, Ton N; Shalek, Alex; Shapiro, Ehud; Sharma, Padmanee; Shin, Jay W; Stegle, Oliver; Stratton, Michael; Stubbington, Michael J T; Theis, Fabian J; Uhlen, Matthias; van Oudenaarden, Alexander; Wagner, Allon; Watt, Fiona; Weissman, Jonathan; Wold, Barbara; Xavier, Ramnik; Yosef, Nir
The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.
Bishop, J.; LaRhette, R.
Evaluating human error or human performance problems and correcting the root causes can help preclude recurrence. The Institute of Nuclear Power Operations (INPO), working with several members and participant utilities in an extended pilot program, has developed a nonpunitive program designed to identify, evaluate, and correct situations that cause human performance errors. The program is called the Human Performance Evaluation System (HPES). Its primary goal is to improve human reliability in overall nuclear plant operations by reducing human error through correction of the conditions that cause the errors. Workers at participating nuclear utilities are encouraged to report their errors and a specially trained plant coordinator investigates and recommends actions to correct the root causes of these errors
Full Text Available During the past ten years expertise in human-computer interaction has shifted from humans interacting with desktop computers to individual human beings or groups of human beings interacting with embedded or mobile technology. Thus, humans are not only interacting with computers but with technology. Obviously, this shift should be reflected in how we educate human-technology interaction (HTI experts today and in the future. We tackle this educational challenge first by analysing current Master’s-level education in collaboration with two universities and second, discussing postgraduate education in the international context. As a result, we identified core studies that should be included in the HTI curriculum. Furthermore, we discuss some practical challenges and new directions for international HTI education.
Luciana Helena Antoniassi da Silva; Fernando Rosado Spilki; Adriana Gut Lopes Riccetto; Emilio Elias Baracat; Clarice Weis Arns
The human respiratory syncytial virus (hRSV) and the human metapneumovírus (hMPV) are main etiological agents of acute respiratory infections (ARI). The ARI is an important cause of childhood morbidity and mortality worldwide. hRSV and hMPV are members of the Paramyxoviridae. They are enveloped, non-segmented viruses, with negative-sense single stranded genomes. Respiratory syncytial virus (hRSV) is the best characterized agent viral of this group, associated with respiratory diseases in...
Inadvertent human intrusion has been an issue for the disposal of solid radioactive waste for many years. This paper discusses proposals for an approach for evaluating the radiological significance of human intrusion as put forward by ICRP with contribution from work at IAEA. The approach focuses on the consequences of the intrusion. Protective actions could, however, include steps to reduce the probability of human intrusion as well as the consequences. (author)
Dougherty, E.M.; Fragola, J.R.
The authors present a treatment of human reliability analysis incorporating an introduction to probabilistic risk assessment for nuclear power generating stations. They treat the subject according to the framework established for general systems theory. Draws upon reliability analysis, psychology, human factors engineering, and statistics, integrating elements of these fields within a systems framework. Provides a history of human reliability analysis, and includes examples of the application of the systems approach
The Human Genome Project is a massive international research project, costing 3 to 5 billion dollars and expected to take 15 years, which will identify the all the genes in the human genome - i.e. the complete sequence of bases in human DNA. The prize will be the ability to identify genes causing or predisposing to disease, and in some cases the development of gene therapy, but this new knowledge will raise important ethical issues
Title: Modern Human Capital Management Seminar date: 30th of May 2008 Course: Master thesis in Business Administration, 15 ECTS Authors: Madita Feldberger Supervisor: Lars Svensson Keywords: Human capital, SWOT Analysis, Strategic Map, Balanced Scorecard Research Problem: Despite of the success of Human Capital Management (HCM) in research it did not arrive yet in the HR departments of many companies. Numerous firms even have problems to set their strategic goals with focus on HR. The HR Bala...
Bauser, M.; Williams, R.
This paper addresses options for dealing with human intrusion in terms of performance requirements and repository siting and design requirements. Options are presented, along with the advantages and disadvantages of certain approaches. At the conclusion, a conceptual approach is offered emphasizing both the minimization of subjective judgements concerning future human activity, and specification of repository requirements to minimize the likelihood of human intrusion and any resulting, harmful effects should intrusion occur
Research Laboratory AFETR Air Force Eastern Test Range AFFTC Air Force Flight Test Center AFHRL Air Force Human Resources Laboratory AFR Air Force...performance requirements through the most effective use of man’s performance capability. 13 Human Engineering is one of five elements in the Human...applied judiciously and tailored to fit * the program or program phase and the acquisition strategy to achieve cost effective acquisition and life cycle
Mitrović Sanja M.; Kranjčić-Zec Ivana F.; Arsić-Arsenijević Valentina S.; Džamić Aleksandar M.; Radonjić Ivana V.
Introduction Babesiosis is caused by intraerythrocytic parasites of the genus Babesia, which is a common animal infection worldwide. This protozoa requires both a competent vertebrate and a nonvertebrate host (Ixodes sp. etc.) to maintain the transmission cycle. Human babesiosis Human babesiosis is predominantly caused by Babesia microti (rodent-borne piroplasm, an emerging zoonosis in humans in North America) and by Babesia divergens (bovine pathogen, in Europe). Occasionally, infection in A...
Joly-Mascheroni, Ramiro M; Senju, Atsushi; Shepherd, Alex J
This study is the first to demonstrate that human yawns are possibly contagious to domestic dogs (Canis familiaris). Twenty-nine dogs observed a human yawning or making control mouth movements. Twenty-one dogs yawned when they observed a human yawning, but control mouth movements did not elicit yawning from any of them. The presence of contagious yawning in dogs suggests that this phenomenon is not specific to primate species and may indicate that dogs possess the capacity for a rudimentary f...
Federal Laboratory Consortium — Biochemistry:Improvements in energy metabolism, muscular strength and endurance capacity have a basis in biochemical and molecular adaptations within the human body....
Nielsen, Jens Bo
Human studies in the past three decades have provided us with an emerging understanding of how cortical and spinal networks collaborate to ensure the vast repertoire of human behaviors. We differ from other animals in having direct cortical connections to spinal motoneurons, which bypass spinal...... the central motor command by opening or closing sensory feedback pathways. In the future, human studies of spinal motor control, in close collaboration with animal studies on the molecular biology of the spinal cord, will continue to document the neural basis for human behavior. Expected final online...
McCarthy, Ellen E
...: To provide an agile, adaptive, integrated, and innovative defense intelligence workforce through a deliberate process identifying, implementing, and directing human capital organizational, doctrinal...
Clarken, Rodney H.
The key to human resource development is in actualizing individual and collective thinking, feeling and choosing potentials related to our minds, hearts and wills respectively. These capacities and faculties must be balanced and regulated according to the standards of truth, love and justice for individual, community and institutional development,…
Reidsma, Dennis; Ruttkay, Z.M.; Huang, T; Nijholt, Antinus; Pantic, Maja; Pentland, A.
The vision of Ambient Intelligence (AmI) presumes a plethora of embedded services and devices that all endeavor to support humans in their daily activities as unobtrusively as possible. Hardware gets distributed throughout the environment, occupying even the fabric of our clothing. The environment
Follmar-Otto, Petra; Rabe, Heike
The first study - "A human rights approach against human trafficking - International obligations and the status of implementation in Germany" - analyses how the prohibition of human trafficking and the resulting state obligations are anchored in human rights. The more recent specialised international agreements on human trafficking and law-making in the European Union are then presented. The emphasis is on the Council of Europe Convention, which professes to treat human trafficking in a human...
de Baets, Antoon; Wright, James
In this article, six basic debates about human rights are clarified from a historical perspective: the origin of human rights as moral rights connected to the natural law doctrine and opposed to positive rights; the wave of criticism of their abstract and absolute character by nineteenth-century
O'Brien, Megan K; Ahmed, Alaa A
It long has been appreciated that humans behave irrationally in economic decisions under risk: they fail to objectively consider uncertainty, costs, and rewards and instead exhibit risk-seeking or risk-averse behavior. We hypothesize that poor estimates of motor variability (influenced by motor task) and distorted probability weighting (influenced by relevant emotional processes) contribute to characteristic irrationality in human movement decisions.
Design approaches are now being applied all over the world as a powerful approach to innovating public policies and services. Christian Bason, author of Leading public design: Discovering human-centred governance, argues that by bringing design methods into play, public managers can lead change...... with citizens at the centre, and discover a new model for steering public organisations: human-centred governance....
Brown, J.; Vos, de W.M.; Distefano, P.S.; Doré, J.; Huttenhower, C.; Knight, R.; Lawley, T.D.; Raes, J.; Turnbaugh, P.
Over the past decade, an explosion of descriptive analyses from initiatives, such as the Human Microbiome Project (HMP) and the MetaHIT project, have begun to delineate the human microbiome. Inhabitants of the intestinal tract, nasal passages, oral cavities, skin, gastrointestinal tract and
The protection of sensitive individuals within a population dictates that measures other than central tendencies be employed to estimate risk. The refinement of human health risk assessments for chemicals metabolized by the liver to reflect data on human variability can be accomplished through (1) the characterization of enzyme expression in large banks of human liver samples, (2) the employment of appropriate techniques for the quantification and extrapolation of metabolic rates derived in vitro, and (3) the judicious application of physiologically based pharmacokinetic (PBPK) modeling. While in vitro measurements of specific biochemical reactions from multiple human samples can yield qualitatively valuable data on human variance, such measures must be put into the perspective of the intact human to yield the most valuable predictions of metabolic differences among humans. For quantitative metabolism data to be the most valuable in risk assessment, they must be tied to human anatomy and physiology, and the impact of their variance evaluated under real exposure scenarios. For chemicals metabolized in the liver, the concentration of parent chemical in the liver represents the substrate concentration in the MichaelisMenten description of metabolism. Metabolic constants derived in vitro may be extrapolated to the intact liver, when appropriate conditions are met. Metabolic capacity Vmax; the maximal rate of the reaction) can be scaled directly to the concentration
Mulenburg, Gerald M. (Inventor); Vernikos, Joan (Inventor)
A human powered centrifuge has independently established turntable angular velocity and human power input. A control system allows excess input power to be stored as electric energy in a battery or dissipated as heat through a resistors. In a mechanical embodiment, the excess power is dissipated in a friction brake.
This book offers a exiting new explanation of human evolution. Based on insight from Anthropology is shows that human became 'cultured' beings capable of symbolic thought by developing rasting kinship based between groups that could not other wise survive in the harah climate condition during...
van den Broek, Egon; Schouten, Th.E.; Kisters, P.M.F.
A unique color space segmentation method is introduced. It is founded on features of human cognition, where 11 color categories are used in processing color. In two experiments, human subjects were asked to categorize color stimuli into these 11 color categories, which resulted in markers for a
Köhler, Julia R; Hube, Bernhard; Puccia, Rosana; Casadevall, Arturo; Perfect, John R
Fungi must meet four criteria to infect humans: growth at human body temperatures, circumvention or penetration of surface barriers, lysis and absorption of tissue, and resistance to immune defenses, including elevated body temperatures. Morphogenesis between small round, detachable cells and long, connected cells is the mechanism by which fungi solve problems of locomotion around or through host barriers. Secretion of lytic enzymes, and uptake systems for the released nutrients, are necessary if a fungus is to nutritionally utilize human tissue. Last, the potent human immune system evolved in the interaction with potential fungal pathogens, so few fungi meet all four conditions for a healthy human host. Paradoxically, the advances of modern medicine have made millions of people newly susceptible to fungal infections by disrupting immune defenses. This article explores how different members of four fungal phyla use different strategies to fulfill the four criteria to infect humans: the Entomophthorales, the Mucorales, the Ascomycota, and the Basidiomycota. Unique traits confer human pathogenic potential on various important members of these phyla: pathogenic Onygenales comprising thermal dimorphs such as Histoplasma and Coccidioides ; the Cryptococcus spp. that infect immunocompromised as well as healthy humans; and important pathogens of immunocompromised patients- Candida , Pneumocystis , and Aspergillus spp. Also discussed are agents of neglected tropical diseases important in global health such as mycetoma and paracoccidiomycosis and common pathogens rarely implicated in serious illness such as dermatophytes. Commensalism is considered, as well as parasitism, in shaping genomes and physiological systems of hosts and fungi during evolution.
Journal Homepage Image. Global Journal of Humanities is aimed at promoting reasearch in all areas of Humanities including philosophy, languages, linguistics, literature, history, fine/applied arts, theater arts, architecture, etc. Visit the Global Journal Series website here: http://www.globaljournalseries.com/ ...
How can one measure the value of teaching the humanities? The problem of assessment and accountability is prominent today, of course, in secondary and higher education. It is perhaps even more acute for those who teach the humanities in nontraditional settings, such as medical and other professional schools. The public assumes that academes can…
Olsen, Peter; Rasmussen, Mads; Zhu, Wei
BACKGROUND: Morphine has been found in cancer cell lines originating from human and animal cells. Thus, it became important to demonstrate whether or not actual tumours contain this opiate alkaloid. MATERIAL/METHODS: Human glioma tissues were biochemically treated to isolate and separate endogenous...
Centering on strategic objective of reform and development,CIAE formulated its objectives in human resource construction for the 13th Five-year Plan period,and achieved new apparent progress in human resource construction in 2015.1 Implementation of"LONGMA Project"
Koopman, Hubertus F.J.M.
The part of (bio)mechanics that studies the interaction of forces on the human skeletal system and its effect on the resulting movement is called rigid body dynamics. Some basic concepts are presented: A mathematical formulation to describe human movement and how this relates on the mechanical loads
Vaupel, James W
Human senescence has been delayed by a decade. This finding, documented in 1994 and bolstered since, is a fundamental discovery about the biology of human ageing, and one with profound implications for individuals, society and the economy. Remarkably, the rate of deterioration with age seems...
Human intestinal spirochaetosis is a condition of the colon that is characterized by the presence of spirochaetes attached to the mucosal cells of the colon. These spirochaetes belong to the family Brachyspiraceae and two species are known to occur in humans: Brachyspira aalborgi and Brachyspira
Iversen, Helle Klingenberg
, which is a human experience. A set-up for investigations of experimental headache and migraine in humans, has been evaluated and headache mechanisms explored by using nitroglycerin and other headache-inducing agents. Nitric oxide (NO) or other parts of the NO activated cascade seems to be responsible...
To strategically manage human capital in education means restructuring the entire human resource system so that schools not only recruit and retain smart and capable individuals, but also manage them in ways that support the strategic directions of the organization. These management practices must be aligned with a district's education improvement…
Some background is given on the field of human factors. The nature of problems with current human/computer interfaces is discussed, some costs are identified, ideal attributes of graceful system interfaces are outlined, and some reasons are indicated why it's not easy to fix the problems
Berman, Howard R.
The international community has developed a system of human rights law relevant to many areas of legal encounter, which American law schools have been slow to incorporate into curricula. Teaching human rights law provides an opportunity for law schools to enrich the learning process and contribute creatively to the respect for rights in society.…
Junior League of Ogden, UT.
This handbook was developed to promote interest in humane education and to encourage the adoption of humane education projects. Although specifically designed to assist Junior Leagues in developing such projects, the content should prove valuable to animal welfare organizations, zoos, aquariums, nature centers, and other project-oriented groups…
When students generate mind maps, or concept maps, the maps are usually on paper, computer screens, or a blackboard. Human Mind Maps require few resources and little preparation. The main requirements are space where students can move around and a little creativity and imagination. Mind maps can be used for a variety of purposes, and Human Mind…
Urban governance on the basis of human rights can help to set up problem solving mechanisms to guarantee social peace, economic growth and political participation.If states both integrate more in international or regional human rights regime and give more autonomy to urban governments and local
Bent Fausing "The Humane Technology", abstract (for The Two Cultures: Balancing Choices and Effects Oxford University July 20-26, 2008). The paper will investigate the use of technology in everyday aesthetics such as TV-commercials for mobile phones for Nokia, which slogan is, as it is well known......, "Nokia - connecting people". Which function does this technology get in narratives, images, interactions and affects here? The mobile phone and its digital camera are depicted as being able to make a unique human presence and interaction. The medium, the technology is a necessary helper to get...... towards this very special and lost humanity. Without the technology, no special humanity is the prophecy. This personification or anthropomorphism is important for the branding of new technology. The technology is seen as creating a technotranscendens towards a more qualified humanity, which is in contact...
Olsen, Peter; Rasmussen, Mads; Zhu, Wei
BACKGROUND: Morphine has been found in cancer cell lines originating from human and animal cells. Thus, it became important to demonstrate whether or not actual tumours contain this opiate alkaloid. MATERIAL/METHODS: Human glioma tissues were biochemically treated to isolate and separate endogeno...... of the solutions used in the study nor was it present as a residual material in blank HPLC runs. CONCLUSIONS: Morphine is present in human gliomas, suggesting that it may exert an action that effects tumour physiology/pathology.......BACKGROUND: Morphine has been found in cancer cell lines originating from human and animal cells. Thus, it became important to demonstrate whether or not actual tumours contain this opiate alkaloid. MATERIAL/METHODS: Human glioma tissues were biochemically treated to isolate and separate endogenous...
Regev, Aviv; Teichmann, Sarah A.; Lander, Eric S.
The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international...... collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells...... in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early...
Full Text Available The paper deals with the structure, mechanisms, practices and perspectives of the Human Rights Council, the UN body that, at universal level is the most important body in this area. Introductory section provides for a brief overview of the origins of human rights and the work of the Commission on Human Rights, in whose jurisdiction were questions of human rights before the establishment of the Council. After the introductory section the author gives an analysis of the structure, objectives, mandate and main procedures for the protection of human rights within the united Nations. In the final section the authorpoints out the advantages of this authority and criticism addressed to it, with emphasis on the possibility and the need for its reform.
Waste is a human concept, referring to things that have no use to human beings and arising entirely from human activities. It is the useless residue of any human process that affects the economy or environment. The changes brought about by the industrial revolution are enormous; fossil fuels, not just photosynthesis, now provide energy and wastes at rates far exceeding the capacity of the ecosystem to absorb or recycle. Three major problems face the Planet: accelerated population growth, accelerated use of resources for energy and industry, and the disproportionate use of resources and waste between the northern and southern parts of the Planet. Knowledge and science are in a position to provide both human creativity and the directed technology to take remedial action and rediscover harmony between nature and mankind. Only social and political will is lacking
Strucic, M.; Kavsek, D.
Human performance remains a significant factor for management attention not only from a reactor safety perspective, but also from a financial one. Recent significant events analysis shows that human errors are still dominant causes and contributors to them. An analysis of significant events in nuclear industry occurred through 15-years period revealed that three of four significant events were triggered by human error, although the number of events have dropped by more than a factor of four. A number of human performance breakdowns occurred in the application of errorprevention techniques. These included a lack of pre-job briefs, inadequate turnover of tasks, ineffective use of peer checking, inadequate procedure adherence, and failure to apply a questioning attitude when unexpected changes were encountered in the task. Attempts by the industry to improve human performance have traditionally focused at the worker level. However, human error occurs within the context of the organization, which can either foster or resist human error. The greatest room for improvement lies not only in the continued improvement of front-line worker performance but more so in the identification and elimination of weaknesses in the organizational and managerial domains that contributes to worker performance at the job site. Based on mentioned analysis, other industrial sources and own operating experience, NPP Krsko is paying more attention to improve human performance among own as well as contractor workers. Through series of programs and activities, such as Reactivity Management Program, Safety Culture Program, Self-assessment Program, Corrective Action Program, Plant Performance Monitoring Program, developed in last few years, and through new procedures, written guides and publications, training and management efforts, number of human errors is going to be reduced. Involvement of higher levels of NPP Krsko organization in promotion and use of Human Performance techniques is
Dischinger, H. Charles, Jr.
The development of models to represent human characteristics and behaviors in human factors is broad and general. The term "model" can refer to any metaphor to represent any aspect of the human; it is generally used in research to mean a mathematical tool for the simulation (often in software, which makes the simulation digital) of some aspect of human performance and for the prediction of future outcomes. This section is restricted to the application of human models in physical design, e.g., in human factors engineering. This design effort is typically human interface design, and the digital models used are anthropometric. That is, they are visual models that are the physical shape of humans and that have the capabilities and constraints of humans of a selected population. They are distinct from the avatars used in the entertainment industry (movies, video games, and the like) in precisely that regard: as models, they are created through the application of data on humans, and they are used to predict human response; body stresses workspaces. DHM enable iterative evaluation of a large number of concepts and support rapid analysis, as compared with use of physical mockups. They can be used to evaluate feasibility of escape of a suited astronaut from a damaged vehicle, before launch or after an abort (England, et al., 2012). Throughout most of human spaceflight, little attention has been paid to worksite design for ground workers. As a result of repeated damage to the Space Shuttle which adversely affected flight safety, DHM analyses of ground assembly and maintenance have been developed over the last five years for the design of new flight systems (Stambolian, 2012, Dischinger and Dunn Jackson, 2014). The intent of these analyses is to assure the design supports the work of the ground crew personnel and thereby protect the launch vehicle. They help the analyst address basic human factors engineering questions: can a worker reach the task site from the work platform
van Keimpema, Martine; Grüneberg, Leonie J; Schilder-Tol, Esther J M; Oud, Monique E C M; Beuling, Esther A; Hensbergen, Paul J; de Jong, Johann; Pals, Steven T; Spaargaren, Marcel
The forkhead transcription factor FOXP1 is generally regarded as an oncogene in activated B cell-like diffuse large B-cell lymphoma. Previous studies have suggested that a small isoform of FOXP1 rather than full-length FOXP1, may possess this oncogenic activity. Corroborating those studies, we herein show that activated B cell-like diffuse large B-cell lymphoma cell lines and primary activated B cell-like diffuse large B-cell lymphoma cells predominantly express a small FOXP1 isoform, and that the 5'-end of the Foxp1 gene is a common insertion site in murine lymphomas in leukemia virus- and transposon-mediated insertional mutagenesis screens. By combined mass spectrometry, (quantative) reverse transcription polymerase chain reaction/sequencing, and small interfering ribonucleic acid-mediated gene silencing, we determined that the small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma lacks the N-terminal 100 amino acids of full-length FOXP1. Aberrant overexpression of this FOXP1 isoform (ΔN100) in primary human B cells revealed its oncogenic capacity; it repressed apoptosis and plasma cell differentiation. However, no difference in potency was found between this small FOXP1 isoform and full-length FOXP1. Furthermore, overexpression of full-length FOXP1 or this small FOXP1 isoform in primary B cells and diffuse large B-cell lymphoma cell lines resulted in similar gene regulation. Taken together, our data indicate that this small FOXP1 isoform and full-length FOXP1 have comparable oncogenic and transcriptional activity in human B cells, suggesting that aberrant expression or overexpression of FOXP1, irrespective of the specific isoform, contributes to lymphomagenesis. These novel insights further enhance the value of FOXP1 for the diagnostics, prognostics, and treatment of diffuse large B-cell lymphoma patients. Copyright© Ferrata Storti Foundation.
Haiden, Nadja; Ziegler, Ekhard E
Human milk banks play an essential role by providing human milk to infants who would otherwise not be able to receive human milk. The largest group of recipients are premature infants who derive very substantial benefits from it. Human milk protects premature infants from necrotizing enterocolitis and from sepsis, two devastating medical conditions. Milk banks collect, screen, store, process, and distribute human milk. Donating women usually nurse their own infants and have a milk supply that exceeds their own infants' needs. Donor women are carefully selected and are screened for HIV-1, HIV-2, human T-cell leukemia virus 1 and 2, hepatitis B, hepatitis C, and syphilis. In the milk bank, handling, storing, processing, pooling, and bacterial screening follow standardized algorithms. Heat treatment of human milk diminishes anti-infective properties, cellular components, growth factors, and nutrients. However, the beneficial effects of donor milk remain significant and donor milk is still highly preferable in comparison to formula. © 2017 S. Karger AG, Basel.
Goodman, P.C.; DiPalo, C.A.
Nuclear power plant safety is dependent upon human performance related to plant operations. To provide improvements in human performance, data collection and assessment play key roles. This paper reports on the Human factors Information System (HFIS) which is designed to meet the needs of the human factors specialists of the United States Nuclear Regulatory Commission. These specialists identify personnel errors and provide guidance designed to prevent such errors. HFIS is a simple and modular system designed for use on a personal computer. It is designed to contain four separate modules that provide information indicative of program or function effectiveness as well as safety-related human performance based on programmatic and performance data. These modules include the Human Factors Status module; the Regulatory Programs module; the Licensee Event Report module; and the Operator Requalification Performance module. Information form these modules can either be used separately or can be combined due to the integrated nature of the system. HFIS has the capability, therefore, to provide insights into those areas of human factors that can reduce the probability of events caused by personnel error at nuclear power plants and promote the health and safety of the public. This information system concept can be applied to other industries as well as the nuclear industry
Williams, Michael A.; Rigamonti, Daniele
Human hydrocephalus is a common medical condition that is characterized by abnormalities in the flow or resorption of cerebrospinal fluid (CSF), resulting in ventricular dilatation. Human hydrocephalus can be classified into two clinical forms, congenital and acquired. Hydrocephalus is one of the complex and multifactorial neurological disorders. A growing body of evidence indicates that genetic factors play a major role in the pathogenesis of hydrocephalus. An understanding of the genetic components and mechanism of this complex disorder may offer us significant insights into the molecular etiology of impaired brain development and an accumulation of the cerebrospinal fluid in cerebral compartments during the pathogenesis of hydrocephalus. Genetic studies in animal models have started to open the way for understanding the underlying pathology of hydrocephalus. At least 43 mutants/loci linked to hereditary hydrocephalus have been identified in animal models and humans. Up to date, 9 genes associated with hydrocephalus have been identified in animal models. In contrast, only one such gene has been identified in humans. Most of known hydrocephalus gene products are the important cytokines, growth factors or related molecules in the cellular signal pathways during early brain development. The current molecular genetic evidence from animal models indicate that in the early development stage, impaired and abnormal brain development caused by abnormal cellular signaling and functioning, all these cellular and developmental events would eventually lead to the congenital hydrocephalus. Owing to our very primitive knowledge of the genetics and molecular pathogenesis of human hydrocephalus, it is difficult to evaluate whether data gained from animal models can be extrapolated to humans. Initiation of a large population genetics study in humans will certainly provide invaluable information about the molecular and cellular etiology and the developmental mechanisms of human
Harvesting energy from the users' muscular power to convert this into electricity is a relatively unknown way to power consumer products. It nevertheless offers surprising opportunities for product designers; human-powered products function independently from regular power infrastructure, are convenient and can be environmentally and economically beneficial. This work provides insight into the knowledge required to design human-powered energy systems in consumer products from a scientific perspective. It shows the developments of human-powered products from the first introduction of the BayGen Freeplay radio in 1995 till current products and provides an overview and analysis of 211 human-powered products currently on the market. Although human power is generally perceived as beneficial for the environment, this thesis shows that achieving environmental benefit is only feasible when the environmental impact of additional materials in the energy conversion system is well balanced with the energy demands of the products functionality. User testing with existing products showed a preference for speeds in the range of 70 to 190 rpm for crank lengths from 32 to 95 mm. The muscular input power varied from 5 to 21 W. The analysis of twenty graduation projects from the Faculty of Industrial Design Engineering in the field of human-powered products, offers an interesting set of additional practice based design recommendations. The knowledge based approach of human power is very powerful to support the design of human-powered products. There is substantial potential for improvements in the domains energy conversion, ergonomics and environment. This makes that human power, when applied properly, is environmentally and economically competitive over a wider range of applications than thought previously.
Full Text Available By opening the field of bioethics followed a new wave of intense debate on the theological, philosophical and legal significance of the concept of human dignity . Exactly ten years ago (December 2003 American bioethicist Ruth Maclin has proposed to divest ourselves of the concept of human dignity because it is vague, useless and redundant and that, without any loss, we can replace it by the ethical principle of personal autonomy. Her article was followed by harsh reactions and opposite views. What is this term in so broad, almost inflationary and opposite use is not a reason to deprive him, but, on the contrary, it shows how important it is and that it should be determined at least outline. As universal values and general concept, the human dignity has no pre-defined and narrow, precise meaning. It is more an evaluation horizon, the guiding principle and regulatory ideas that must constantly define and codify by many guaranted human rights and fundamental freedoms. As generic notion of each reasonable law, it is their foundation and a common denominator, legitimising basis of natural but also of positive law. As intrinsic and static value which means the humaneness, the humanity it is absolute, inherent to every human being without distinction and conditioning, as a unique and unrepeatable creation. In this meaning, the dignity is the obligation and limitation of the state, society and each of us. As an ethical and dynamic category, it is not given to us, but it is assign to us, and it is not in us, but always before us, as a guide of our actions in accordance with virtues, to treat ourselves, each other and the nature in a human way. The century in which we live is named the century of molecular biology and genetic engineering because of the enormous potential but also risks to human dignity. Because of that human dignity has become a central principle in all international documents relating to the human genome, genetics and bioethics, adopted
Block, S. [The MITRE Corporation, McLean, VA (US). JASON Program Office; Cornwall, J. [The MITRE Corporation, McLean, VA (US). JASON Program Office; Dally, W. [The MITRE Corporation, McLean, VA (US). JASON Program Office; Dyson, F. [The MITRE Corporation, McLean, VA (US). JASON Program Office; Fortson, N. [The MITRE Corporation, McLean, VA (US). JASON Program Office; Joyce, G. [The MITRE Corporation, McLean, VA (US). JASON Program Office; Kimble, H. J. [The MITRE Corporation, McLean, VA (US). JASON Program Office; Lewis, N. [The MITRE Corporation, McLean, VA (US). JASON Program Office; Max, C. [The MITRE Corporation, McLean, VA (US). JASON Program Office; Prince, T. [The MITRE Corporation, McLean, VA (US). JASON Program Office; Schwitters, R. [The MITRE Corporation, McLean, VA (US). JASON Program Office; Weinberger, P. [The MITRE Corporation, McLean, VA (US). JASON Program Office; Woodin, W. H. [The MITRE Corporation, McLean, VA (US). JASON Program Office
The study reviews Department of Energy supported aspects of the United States Human Genome Project, the joint National Institutes of Health/Department of Energy program to characterize all human genetic material, to discover the set of human genes, and to render them accessible for further biological study. The study concentrates on issues of technology, quality assurance/control, and informatics relevant to current effort on the genome project and needs beyond it. Recommendations are presented on areas of the genome program that are of particular interest to and supported by the Department of Energy.
Minshall, Clare; Nadal, Jodie; Exley, Christopher
It is of burgeoning importance that the human body burden of aluminium is understood and is measured. There are surprisingly few data to describe human excretion of systemic aluminium and almost no reliable data which relate to aluminium in sweat. We have measured the aluminium content of sweat in 20 healthy volunteers following mild exercise. The concentration of aluminium ranged from 329 to 5329μg/L. These data equate to a daily excretion of between 234 and 7192μg aluminium and they strongly suggest that perspiration is the major route of excretion of systemic aluminium in humans. Copyright © 2013 Elsevier GmbH. All rights reserved.
Human activities have circumvented the efficient geochemical cycling of aluminium within the lithosphere and therewith opened a door, which was previously only ajar, onto the biotic cycle to instigate and promote the accumulation of aluminium in biota and especially humans. Neither these relatively recent activities nor the entry of aluminium into the living cycle are showing any signs of abating and it is thus now imperative that we understand as fully as possible how humans are exposed to aluminium and the future consequences of a burgeoning exposure and body burden. The aluminium age is upon us and there is now an urgent need to understand how to live safely and effectively with aluminium.
Broor, Shobha; Bharaj, Preeti
Pneumovirus infection remains a significant problem for both human and veterinary medicine. Both avian pneumovirus (aMPV, Turkey rhinotracheitis virus) and human metapneumovirus (hMPV) are pathogens of birds and humans, which are associated with respiratory tract infections. Based on their different genomic organization and low level of nucleotide (nt) and amino acid (aa) identity with paramyxoviruses in the genus Pneumovirus, aMPV and hMPV have been classified into a new genus referred to as Metapneumovirus. The advancement of our understanding of pneumovirus biology and pathogenesis of pneumovirus disease in specific natural hosts can provide us with strategies for vaccine formulations and combined antiviral and immunomodulatory therapies.
... chromosomes and plasmids. Transposons often carry genes specifying antimicrobial resistance. Virus An extremely small infective agent, visible only with an electron microscope. Viruses can cause disease in humans, animals and plants. Viruses consist of a protein coat ...
Sep 1, 2009 ... germline stem cell maintenance, epigenetic regulation, and transposition. Although the ... Species. MILI. (PIWIL2 In human. Transposon, and piRNA clusters. 24-28 nts .... However, the differences among embryonic, neonatal,.
Skibsbye, Lasse; Jespersen, Thomas; Christ, Torsten
BACKGROUND: Refractoriness of cardiac cells limits maximum frequency of electrical activity and protects the heart from tonic contractions. Short refractory periods support major arrhythmogenic substrates and augmentation of refractoriness is therefore seen as a main mechanism of antiarrhythmic...... drugs. Cardiomyocyte excitability depends on availability of sodium channels, which involves both time- and voltage-dependent recovery from inactivation. This study therefore aims to characterise how sodium channel inactivation affects refractoriness in human atria. METHODS AND RESULTS: Steady......-state activation and inactivation parameters of sodium channels measured in vitro in isolated human atrial cardiomyocytes were used to parameterise a mathematical human atrial cell model. Action potential data were acquired from human atrial trabeculae of patients in either sinus rhythm or chronic atrial...
This presentation covers the high points of the Human Reliability Program, including certification/decertification, critical positions, due process, organizational structure, program components, personnel security, an overview of the US DOE reliability program, retirees and academia, and security program integration.
... factors for developing them, such as taking oral contraceptives . A safety review of Gardasil in Denmark and ... and venous thromboembolic adverse events after immunisation of adolescent girls with quadrivalent human papillomavirus vaccine in Denmark ...
Farbrot, J.E.; Nihlwing, Ch.; Svengren, H.
New requirements for enhanced safety and design changes in process systems often leads to a step-wise installation of new information and control equipment in the control room of older nuclear power plants, where nowadays modern digital I and C solutions with screen-based human-machine interfaces (HMI) most often are introduced. Human factors (HF) expertise is then required to assist in specifying a unified, integrated HMI, where the entire integration of information is addressed to ensure an optimal and effective interplay between human (operators) and machine (process). Following a controlled design process is the best insurance for ending up with good solutions. This paper addresses the approach taken when introducing modern human-machine communication in the Oskarshamn 1 NPP, the results, and the lessons learned from this work with high operator involvement seen from an HF point of view. Examples of possibilities modern technology might offer for the operators are also addressed. (orig.)
Modern dexterous communication technology is progressively enabling humans to communicate their information through them with speech (aural) and media (optical) as underpinning essence. Humans realize this kind of aural and optical information by their optical and auditory senses. However, due...... to certain constraints, the ability to incorporate the other three sensory features namely, olfactory, gustatory, and tactile are still far from reality. Human bond communication is a novel concept that incorporates olfactory, gustatory, and tactile that will allow more expressive and holistic sensory...... information exchange through communication techniques for more human sentiment centric communication. This concept endorses the need of inclusion of other three senses and proposes an innovative approach of holistic communication for future communication network....
OAS, including its organizational structure, each organizational unit's staffing, vacant posts, and a list of procurement notices for formal bids, links to the performance contract and travel control Plan Human Resources Organizational Structure Functions of each organizational unit Vacant Posts
Spaceflight is challenging. Human spaceflight is far more challenging,.Those familiar with spaceflight recognize that human spaceflight is more than tacking an environmental control system on an existing spacecraft, that there are a number of serious technical challenges involved in sending people out into space and bringing them back home safely.The return trip, bringing the crew back to the surface of the earth safely, is more than just an additional task, it's the new imperative. Differences between manned and unmanned spaceflight are more than technical. The human element forces a change in philosophy, a mindset that will likely touch every aspect of flight from launch through mission and return. Seasoned space professionals used to the paradigms and priorities of unmanned flight need to be cognizant of these differences and some of the implications, perhaps most especially because mission success and human safety priorities are sometimes contradictory.
Full Text Available Human dignity has become a major moral directive in the contemporary ethical reflection on human rights and bio-ethics. This article examines the theological foundations laid by the reformer Calvin regarding the inherent dignity of people, and his influence on post-World War ethical reflection about the violations of human rights. In this article his views on the “imago dei” and common grace, the “lex naturae” and the obligations of the civil authority are investigated in order to illuminate his ideas about the dignity of human beings. The article then deals with the influence of these ideas in the influential works of the twentieth century’s reformed theologians Barth, Berkhouwer and Moltmann.
and the design process, in ethical and society-related concerns, and in evaluating how designs fulfill needs and solve problems. Designing Human Technologies subscribes to a broad technology concept including information and communication, mobile, environmental/sustainable and energy technologies......Design is increasingly becoming a part of the university curriculum and research agenda. The keynote present and discuss Designing Human Technologies – an initiative aiming at establishing a design oriented main subject area alongside traditional main subject areas such as Natural Science......, the Humanities, and Social Science. The initiative broadens the perspective of IS and recognize reflections on aesthetics, ethics, values, connections to politics, and strategies for enabling a better future as legitimate parts of the research agenda. Designing Human Technologies is a design-oriented Strategic...
... cryosections are associated with anatomical terminology. AnatLine : A prototype system consisting of an anatomical image database and ... further information is available Publications VHJOE: Visible Human Journal of Endoscopy. NLM's Current Bibliographies in Medicine, Visible ...
Full Text Available In this paper the authors analyze the process of negotiating and beginning of the United Nations Declaration on Human Cloning as well as the paragraphs of the very Declaration. The negotiation was originally conceived as a clear bioethical debate that should have led to a general agreement to ban human cloning. However, more often it had been discussed about human rights, cultural, civil and religious differences between people and about priorities in case of eventual conflicts between different value systems. In the end, a non-binding Declaration on Human Cloning had been adopted, full of numerous compromises and ambiguous formulations, that relativized the original intention of proposer states. According to authors, it would have been better if bioethical discussion and eventual regulations on cloning mentioned in the following text had been left over to certain professional bodies, and only after the public had been fully informed about it should relevant supranational organizations have taken that into consideration.
National Aeronautics and Space Administration — Strategically, the HRP conducts research and technology development that: 1) enables the development or modification of Agency-level human health and performance...
Humanistic behaviorism may provide the necessary bridge between behaviorism and humanism. Perhaps the most humanistic approach to teaching is to learn how certain changes will help students and how these changes can be accomplished. (Author/MLF)
Author argues that humanism and behaviorism are not necessarily exclusive of one another, and that principles of behaviorism, when thoughtfully applied, can lead to the achievement of humanistic goals. (RW)
Salas, Eduardo; Maurino, Daniel E
.... HFA offers a comprehensive overview of the topic, taking readers from the general to the specific, first covering broad issues, then the more specific topics of pilot performance, human factors...
Department of Transportation — AVS is now required to collect, track, and report on data from the following Flight, Business and Workforce Plan. The Human Resource Management’s Performance Target...
Federal Laboratory Consortium — Purpose: The purpose of the Human Factors Laboratory is to further the understanding of highway user needs so that those needs can be incorporated in roadway design,...
... into human evolution and origins and serving as a springboard for important medical research. It also addresses issues of confidentiality and individual privacy for participants in genetic diversity research studies.
Feuillet-Le Mintier, B
Biotechnology permits our world to progress. It's a tool to better apprehend the human being, but as well to let him go ahead. Applied to the living, biotechnologies present the same finality. But since their matter concerns effectively the living, they are the sources of specific dangers and particularly of that one to use the improvements obtained on the human to modify the human species. The right of the persons has to find its place to avoid that the fundamental rights of the human personality shall undergo harm. This mission assigned to the right of the persons is as so much invaluable that the economical stakes are particularly important in the domain of the biotechnologies.
performance factors that affect the ability of a human to drive at night, which includes the eyesight of the driver, the fatigue level of the driver...where human factors are factors that affect the performance of an individual. 7 for human interaction. For instance, they explain the various human... affecting trust in human-robot interaction. Human Factors 53(5), 517-527 (2001) 35. Hart, S. G. and Staveland, L. E. Development of NASA-TLX (Task
CALTON,TERRI L.; PETERS,RALPH R.
Automatic assembly sequencing and visualization tools are valuable in determining the best assembly sequences, but without Human Factors and Figure Models (HFFMs) it is difficult to evaluate or visualize human interaction. In industry, accelerating technological advances and shorter market windows have forced companies to turn to an agile manufacturing paradigm. This trend has promoted computerized automation of product design and manufacturing processes, such as automated assembly planning. However, all automated assembly planning software tools assume that the individual components fly into their assembled configuration and generate what appear to be a perfectly valid operations, but in reality the operations cannot physically be carried out by a human. Similarly, human figure modeling algorithms may indicate that assembly operations are not feasible and consequently force design modifications; however, if they had the capability to quickly generate alternative assembly sequences, they might have identified a feasible solution. To solve this problem HFFMs must be integrated with automated assembly planning to allow engineers to verify that assembly operations are possible and to see ways to make the designs even better. Factories will very likely put humans and robots together in cooperative environments to meet the demands for customized products, for purposes including robotic and automated assembly. For robots to work harmoniously within an integrated environment with humans the robots must have cooperative operational skills. For example, in a human only environment, humans may tolerate collisions with one another if they did not cause much pain. This level of tolerance may or may not apply to robot-human environments. Humans expect that robots will be able to operate and navigate in their environments without collisions or interference. The ability to accomplish this is linked to the sensing capabilities available. Current work in the field of cooperative
With human colonization of Mars, I think you will see a higher standard of civilization, just as America set a higher standard of civilization which then promulgated back into Europe. I think that if you want to maximize human potential, you need a higher standard of civilization, and that becomes an example that benefits everyone. Without an open frontier, closed world ideologies, such as the Malthus Theory, tend to come to the forefront. It is that there are limited resources; therefore, we are all in deadly competition with each other for the limited pot. The result is tyrannical and potentially genocidal regimes, and we've already seen this in the twentieth century. There s no truth in the Malthus Theory, because human beings are the creators of their resources. With every mouth comes a pair of hands and a brain. But if it seems to be true, you have a vector in this direction, and it is extremely unfortunate. It is only in a universe of infinite resources that all humans can be brothers and sisters. The fundamental question which affects humanity s sense of itself is whether the world is changeable or fixed. Are we the makers of our world or just its inhabitants? Some people have a view that they re living at the end of history within a world that s already defined, and there is no fundamental purpose to human life because there is nothing humans can do that matters. On the other hand, if humans understand their own role as the creators of their world, that s a much more healthy point of view. It raises the dignity of humans. Indeed, if we do establish a new branch of human civilization on Mars that grows in time and potency to the point where it cannot really settle Mars, but transforms Mars, and brings life to Mars, we will prove to everyone and for all time the precious and positive nature of the human species and every member of it.
This article analyses the United Nations (UN) Guidelines on Business and Human Rights adopted in 2011 by the UN Human Rights Council from the perspective of transnational business governance interactions (TBGI) analytical framework.1 The article identifies and discusses dimensions of interaction...... in several areas of relevance to transnational business governance interaction and indicates the relevance of the TBGI approach to public regulatory transnational business governance initiatives. The analysis of the Guiding Principles as interactional transnational business governance suggests that this form...
Quality of products and services is seen as a necessity in our modern world. Quality also has important cross-links to safety in our society. It is however suggested, that human beings are living in their industrial environment under the stress of a fractured personality with anxieties and frustrations. Some cultural comparisons with other industrial nations are given. Quality control tailored to human nature is recommended.
Kels, Barry D; Grzybowski, Andrzej; Grant-Kels, Jane M
We review the normal anatomy of the human globe, eyelids, and lacrimal system. This contribution explores both the form and function of numerous anatomic features of the human ocular system, which are vital to a comprehensive understanding of the pathophysiology of many oculocutaneous diseases. The review concludes with a reference glossary of selective ophthalmologic terms that are relevant to a thorough understanding of many oculocutaneous disease processes. Copyright © 2015 Elsevier Inc. All rights reserved.
Ormond, Kelly E; Mortlock, Douglas P; Scholes, Derek T; Bombard, Yvonne; Brody, Lawrence C; Faucett, W Andrew; Garrison, Nanibaa' A; Hercher, Laura; Isasi, Rosario; Middleton, Anna; Musunuru, Kiran; Shriner, Daniel; Virani, Alice; Young, Caroline E
With CRISPR/Cas9 and other genome-editing technologies, successful somatic and germline genome editing are becoming feasible. To respond, an American Society of Human Genetics (ASHG) workgroup developed this position statement, which was approved by the ASHG Board in March 2017. The workgroup included representatives from the UK Association of Genetic Nurses and Counsellors, Canadian Association of Genetic Counsellors, International Genetic Epidemiology Society, and US National Society of Genetic Counselors. These groups, as well as the American Society for Reproductive Medicine, Asia Pacific Society of Human Genetics, British Society for Genetic Medicine, Human Genetics Society of Australasia, Professional Society of Genetic Counselors in Asia, and Southern African Society for Human Genetics, endorsed the final statement. The statement includes the following positions. (1) At this time, given the nature and number of unanswered scientific, ethical, and policy questions, it is inappropriate to perform germline gene editing that culminates in human pregnancy. (2) Currently, there is no reason to prohibit in vitro germline genome editing on human embryos and gametes, with appropriate oversight and consent from donors, to facilitate research on the possible future clinical applications of gene editing. There should be no prohibition on making public funds available to support this research. (3) Future clinical application of human germline genome editing should not proceed unless, at a minimum, there is (a) a compelling medical rationale, (b) an evidence base that supports its clinical use, (c) an ethical justification, and (d) a transparent public process to solicit and incorporate stakeholder input. Copyright © 2017 American Society of Human Genetics. All rights reserved.
Epidemiologic studies conducted in the past 30 years to investigate the protective functions of human milk strongly support the notion that breastfeeding prevents infantile infections, particularly those affecting the gastrointestinal and respiratory tracts. However, more recent clinical and experimental observations also suggest that human milk not only provides passive protection, but also can directly modulate the immunological development of the recipient infant. The study of this remarkable defense system in human milk has been difficult because of its biochemical complexity, the small concentration of certain bioactive components, the compartmentalization of some of these agents, the dynamic quantitative and qualitative changes of milk during lactation, and the lack of specific reagents to quantify these agents. However, a host of bioactive substances, including hormones, growth factors, and immunological factors such as cytokines, have been identified in human milk. Cytokines are pluripotent polypeptides that act in autocrine/paracrine fashions by binding to specific cellular receptors. They operate in networks and orchestrate the development and functions of immune system. Several different cytokines and chemokines have been discovered in human milk in the past years, and the list is growing very rapidly. This article will review the current knowledge about the increasingly complex network of chemoattractants, activators, and anti-inflammatory cytokines present in human milk and their potential role in compensating for the developmental delay of the neonate immune system. Copyright 2010. Published by Mosby, Inc.
Hardwick, R.J. Jr.
Operating nuclear power plants requires high standards of performance, extensive training and responsive management. Despite our best efforts inappropriate human actions do occur, but they can be managed. An extensive review of License Event Reports (LERs) was conducted which indicated continual inadequacy in human performance and in evaluation of root causes. Of some 31,000 LERs, about 5,000 or 16% were directly attributable to inappropriate actions. A recent analysis of 87 Significant Event Reports (issued by INPO in 1983) identified inappropriate actions as being the most frequent root cause (44% of the total). A more recent analysis of SERs issued in 1983 and 1984 indicate that 52% of the root causes were attributed to human performance. The Human Performance Evaluation System (HPES) is a comprehensive, coordinated utility/industry system for evaluating and reporting human performance situtations. HPES is a result of the realization that current reporting system provide limited treatment of human performance and rarely provide adequate information about root causes of inappropriate actions by individuals. The HPES was implemented to identify and eliminate root causes of inappropriate actions
Paramore, B.; Peterson, L.R.
''Human Factors'' is concerned with the incorporation of human user considerations into a system in order to maximize human reliability and reduce errors. This Review Plan is intended to assist in the assessment of human factors conditions in existing DOE facilities. In addition to specifying assessment methodologies, the plan describes techniques for improving conditions which are found to not adequately support reliable human performance. The following topics are addressed: (1) selection of areas for review describes techniques for needs assessment to assist in selecting and prioritizing areas for review; (2) human factors engineering review is concerned with optimizing the interfaces between people and equipment and people and their work environment; (3) procedures review evaluates completeness and accuracy of procedures, as well as their usability and management; (4) organizational interface review is concerned with communication and coordination between all levels of an organization; and (5) training review evaluates training program criteria such as those involving: trainee selection, qualification of training staff, content and conduct of training, requalification training, and program management
Paramore, B.; Peterson, L.R. (eds.)
''Human Factors'' is concerned with the incorporation of human user considerations into a system in order to maximize human reliability and reduce errors. This Review Plan is intended to assist in the assessment of human factors conditions in existing DOE facilities. In addition to specifying assessment methodologies, the plan describes techniques for improving conditions which are found to not adequately support reliable human performance. The following topics are addressed: (1) selection of areas for review describes techniques for needs assessment to assist in selecting and prioritizing areas for review; (2) human factors engineering review is concerned with optimizing the interfaces between people and equipment and people and their work environment; (3) procedures review evaluates completeness and accuracy of procedures, as well as their usability and management; (4) organizational interface review is concerned with communication and coordination between all levels of an organization; and (5) training review evaluates training program criteria such as those involving: trainee selection, qualification of training staff, content and conduct of training, requalification training, and program management.
Full Text Available From an initial project to investigate the relationship between magic and traditional medicine as practiced by shamans in Southern rural Asia, the focus of attention gradually shifted to an awareness of the negotiation traditional medicine people or shamans exert between the human community and the larger community of beings. This attentiveness to a more-than-human world does not occur at a supernatural domain above nature or inside her personal self but is the result of the shaman’s special ability to project her consciousness horizontally to other forms of sensibility with which human existence is interwoven. The ecological function of the shaman is to maintain a constant balance between what is taken and what is given from the human community to the larger community. The spirits of indigenous cultures are not defined in opposition to materiality but are essentially those modes of intelligence or awareness that do not possess a human form. By exploring different landscapes, and the sensibility living in them, a new sensitivity is awoken that allows for communication with those intelligences. However, the drowning of these other voices in Western culture, which reduces otherness to an object, creates an uneasiness that is hardly perceived except as an inability to interact with anything more-than-human and its dire consequences in the form of “civilization’s” destructive behavior.
Full Text Available If one compares the articles of the "Universal Declaration of Human Rights" dated December 10th, 1948, with the regulations of the book of Deuteronomy, one detects a surprising abundance of correspondences, or at least of similar tendencies, between them. As the social theorists of the seventeenth and eighteenth centuries, the architects of the catalogue of Human Rights, knew the Scripture very well. References to Deuteronomy are historically well probable and factually hardly coincidental. Deuteronomy rightly boasts about its social laws (4:8 that are unique in the Ancient Near East. The paper orientates itself to the short formula of Human Rights and at the same time to the normative basic character of each human right, as it is formulated in the first article of the declaration: "liberty", "equality", "fraternity". Each of these basic categories are concretised in terms of several Deuteronomic regulations and prove themselves to be central matters of concern within the YHWH religion. Finally, it is outlined how the connection between Deuteronomy and modem expressions of human rights might be explained, and further it is shown what actually makes up the peculiarity of biblical thinking on human rights.
Sumaya, Jennifer Boyer
This slide presentation reviews the work of the Habitability and Human Factors Branch in support of human space flight in two main areas: Applied support to major space programs, and Space research. The field of Human Factors applies knowledge of human characteristics for the design of safer, more effective, and more efficient systems. This work is in several areas of the human space program: (1) Human-System Integration (HSI), (2) Orion Crew Exploration Vehicle, (3) Extravehicular Activity (EVA), (4) Lunar Surface Systems, (5) International Space Station (ISS), and (6) Human Research Program (HRP). After detailing the work done in these areas, the facilities that are available for human factors work are shown.
Bites - human - self-care ... Human bites can occur in 2 ways: If someone bites you If your hand comes into contact ... bite to express anger or other negative feelings. Human bites may be more dangerous than animal bites. ...
Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna
for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps of the humanization experiment protocol. AVAILABILITY: http
Modern human rights instruments ground human rights in the concept of human dignity, without providing an underlying theory of human dignity. This paper examines the central importance of human dignity, understood as not humiliating people, in traditional Jewish ethics. It employs this conception of human dignity to examine and criticize U.S. use of humiliation tactics and torture in the interrogation of terrorism suspects.
Contents: What is human smuggling?; How can we know about human smuggling?; Human smuggling as a migration phenomenon; Human smuggling as a business; The social organizing of human smuggling; Fighting against human smuggling.
Graells, Robert Casals i.; Sibilla, Anna; Bohle, Martin
Anthropogenic global change is a composite process. It consists of societal processes (in the 'noosphere') and natural processes (in the 'bio-geosphere'). The 'noosphere' is the ensemble of social, cultural or political insights ('shared subjective mental concepts') of people. Understanding the composite of societal and natural processes ('human geo-biosphere intersections'), which shapes the features of anthropogenic global change, would benefit from a description that draws equally on natural sciences, social sciences and humanities. To that end it is suggested to develop a concept of 'geo-humanities': This essay presents some aspects of its scope, discussing "knowledge that is to manage", "intentions that are to shape", "choices that are to justify" and "complexity that is to handle". Managing knowledge: That people understand anthropogenic global change requires their insights into how 'human geosphere intersections' function. Insights are formed ('processed') in the noosphere by means of interactions between people. Understanding how 'human geosphere intersections' functions combines scientific, engineering and economic studies with studies of the dynamics of the noosphere. Shaping intentions: During the last century anthropogenic global change developed as the collateral outcome of humankind's accumulated actions. It is caused by the number of people, the patterns of their consumption of resources, and the alterations of their environments. Nowadays, anthropogenic global chance is either an intentional negligence or a conscious act. Justifying choices: Humanity has alternatives how to alter Earth at planetary scale consciously. For example, there is a choice to alter the geo-biosphere or to adjust the noosphere. Whatever the choice, it will depend on people's world-views, cultures and preferences. Thus beyond issues whether science and technology are 'sound' overarching societal issues are to tackle, such as: (i) how to appropriate and distribute natural
Psychogios, Nikolaos; Hau, David D.; Peng, Jun; Guo, An Chi; Mandal, Rupasri; Bouatra, Souhaila; Sinelnikov, Igor; Krishnamurthy, Ramanarayan; Eisner, Roman; Gautam, Bijaya; Young, Nelson; Xia, Jianguo; Knox, Craig; Dong, Edison; Huang, Paul; Hollander, Zsuzsanna; Pedersen, Theresa L.; Smith, Steven R.; Bamforth, Fiona; Greiner, Russ; McManus, Bruce; Newman, John W.; Goodfriend, Theodore; Wishart, David S.
Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at http://www.serummetabolome.ca. PMID:21359215
Trager, E. A.
The purpose of this paper is to describe several aspects of a Human Performance Event Database (HPED) that is being developed by the Nuclear Regulatory Commission. These include the background, the database structure and basis for the structure, the process for coding and entering event records, the results of preliminary analyses of information in the database, and plans for the future. In 1992, the Office for Analysis and Evaluation of Operational Data (AEOD) within the NRC decided to develop a database for information on human performance during operating events. The database was needed to help classify and categorize the information to help feedback operating experience information to licensees and others. An NRC interoffice working group prepared a list of human performance information that should be reported for events and the list was based on the Human Performance Investigation Process (HPIP) that had been developed by the NRC as an aid in investigating events. The structure of the HPED was based on that list. The HPED currently includes data on events described in augmented inspection team (AIT) and incident investigation team (IIT) reports from 1990 through 1996, AEOD human performance studies from 1990 through 1993, recent NRR special team inspections, and licensee event reports (LERs) that were prepared for the events. (author)
Full Text Available So far our open access publishing company MDPI (Multidisciplinary Digital Publishing Institute has published mainly science, medicine and technology journals. To become a multidisciplinary publisher, we launched the journal Sustainability . More recently, we started to run several social science journals, including Societies , Religions , Administrative Sciences  and Behavioral Sciences . Today we published the first paper  of the inaugural issue of Humanities (ISSN 2076-0787. This will be an international open access journal, publishing scholarly papers of high quality across all humanities disciplines. As a publisher, I would like to publish journals surrounding the topics of sustainability and I believe the humanities as a discipline of academic studies are very important. As a scientist, I believed science and technology will only benefit human beings. I was raised in a small village, living a very primitive life in a peasant family: no electricity, no machines, of course no TV and no refrigerator. Now, the life of my children is completely different. Even my own life has completely changed. I have witnessed very rapid changes: more and more machines are used to consume mineral resources and energy and to pollute the environment, in order to produce more and more powerful machines (we are also launching a journal titled Machines, in which the relationship between Man and machine should be an interesting topic.. Machines are more and more like human individuals consuming resources themselves (we are launching a journal titled Resources. [...
Manrique, Pilar; Bolduc, Benjamin; Walk, Seth T; van der Oost, John; de Vos, Willem M; Young, Mark J
The role of bacteriophages in influencing the structure and function of the healthy human gut microbiome is unknown. With few exceptions, previous studies have found a high level of heterogeneity in bacteriophages from healthy individuals. To better estimate and identify the shared phageome of humans, we analyzed a deep DNA sequence dataset of active bacteriophages and available metagenomic datasets of the gut bacteriophage community from healthy individuals. We found 23 shared bacteriophages in more than one-half of 64 healthy individuals from around the world. These shared bacteriophages were found in a significantly smaller percentage of individuals with gastrointestinal/irritable bowel disease. A network analysis identified 44 bacteriophage groups of which 9 (20%) were shared in more than one-half of all 64 individuals. These results provide strong evidence of a healthy gut phageome (HGP) in humans. The bacteriophage community in the human gut is a mixture of three classes: a set of core bacteriophages shared among more than one-half of all people, a common set of bacteriophages found in 20-50% of individuals, and a set of bacteriophages that are either rarely shared or unique to a person. We propose that the core and common bacteriophage communities are globally distributed and comprise the HGP, which plays an important role in maintaining gut microbiome structure/function and thereby contributes significantly to human health.
Tiebout, R.F.; van Boxtel-Oosterhof, F.; Stricker, E.A.M.; Zeijlemaker, W.P.
Hybrid hybridomas are obtained by fusion of two cells, each producing its own antibody. Several authors have reported the construction of murine hybrid hybridomas with the aim to obtain bispecific monoclonal antibodies. The authors have investigated, in a model system, the feasibility of constructing a human hybrid hybridoma. They fused two monoclonal cell lines: an ouabain-sensitive and azaserine/hypoxanthine-resistant Epstein-Barr virus-transformed human cell line that produces an IgG1kappa antibody directed against tetanus toxiod and an azaserine/hypoxanthine-sensitive and ouabain-resistant human-mouse xenohybrid cell line that produces a human IgG1lambda antibody directed against hepatitis-B surface antigen. Hybrid hybridoma cells were selected in culture medium containing azaserine/hypoxanthine and ouabain. The hybrid nature of the secreted antibodies was analyzed by means of two antigen-specific immunoassay. The results show that it is possible, with the combined use of transformation and xenohybridization techniques, to construct human hybrid hybridomas that produce bispecific antibodies. Bispecific antibodies activity was measured by means of two radioimmunoassays.
Liao, Matthew S
What makes something a human right? What is the relationship between the moral foundations of human rights and human rights law? What are the difficulties of appealing to human rights? This book offers the first comprehensive survey of current thinking on the philosophical foundations of human rights. Divided into four parts, this book focusses firstly on the moral grounds of human rights, for example in our dignity, agency, interests or needs. 'Secondly, it looks at the implications that different moral perspectives on human rights bear for human rights law and politics. Thirdly, it discusses specific and topical human rights including freedom of expression and religion, security, health and more controversial rights such as a human right to subsistence. The final part discusses nuanced critical and reformative views on human rights from feminist, Kantian and relativist perspectives among others. The essays represent new and canonical research by leading scholars in the field. Each part is comprised of a set...
Schubö, Anna; Vesper, Cordula; Wiesbeck, Mathey
and describing human-human interaction in terms of goal-oriented movement coordination is considered an important and necessary step for designing and describing human-robot interaction. In the present scenario, trajectories of hand and finger movements were recorded while two human participants performed......The present paper describes a scenario for examining mechanisms of movement coordination in humans and robots. It is assumed that coordination can best be achieved when behavioral rules that shape movement execution in humans are also considered for human-robot interaction. Investigating...... coordination were affected. Implications for human-robot interaction are discussed....
Suzana Miovska Spaseva
Full Text Available The article examines the complex role and great responsibility of the education today in development of the moral strength and human values of the children and youth. At the beginning of the article the author reconsiders the pedagogical ideas of Maria Montessori and her concept of education for peace as an instrument for reconstruction of the society and for improvement of the human living. Than the analysis of the moral values in the contemporary society is made and several issues and dilemmas are discussed referring the value disorientation of the youth and the importance of the models of adult’s moral behavior in their search for personal identity. On the basis of this analysis, the human dimension of the education is elaborated enhancing the need for its understanding as support of development, which is based on several crucial elements: love, freedom and spirit of community.
Brown, Emma S; Allsopp, Philip J; Magee, Pamela J; Gill, Chris I R; Nitecki, Sonja; Strain, Conall R; McSorley, Emeir M
Seaweeds may have an important role in modulating chronic disease. Rich in unique bioactive compounds not present in terrestrial food sources, including different proteins (lectins, phycobiliproteins, peptides, and amino acids), polyphenols, and polysaccharides, seaweeds are a novel source of compounds with potential to be exploited in human health applications. Purported benefits include antiviral, anticancer, and anticoagulant properties as well as the ability to modulate gut health and risk factors for obesity and diabetes. Though the majority of studies have been performed in cell and animal models, there is evidence of the beneficial effect of seaweed and seaweed components on markers of human health and disease status. This review is the first to critically evaluate these human studies, aiming to draw attention to gaps in current knowledge, which will aid the planning and implementation of future studies.
Taboureau, Olivier; Audouze, Karine
During the past decades, many epidemiological, toxicological and biological studies have been performed to assess the role of environmental chemicals as potential toxicants for diverse human disorders. However, the relationships between diseases based on chemical exposure have been rarely studied...... by computational biology. We developed a human environmental disease network (EDN) to explore and suggest novel disease-disease and chemical-disease relationships. The presented scored EDN model is built upon the integration on systems biology and chemical toxicology using chemical contaminants information...... and their disease relationships from the reported TDDB database. The resulting human EDN takes into consideration the level of evidence of the toxicant-disease relationships allowing including some degrees of significance in the disease-disease associations. Such network can be used to identify uncharacterized...
This paper deals with the problem of designing more humanised computer systems. This problem can be formally described as the need for defining human design criteria, which — if used in the design process - will secure that the systems designed get the relevant qualities. That is not only...... the necessary functional qualities but also the needed human qualities. The author's main argument is, that the design process should be a dialectical synthesis of the two points of view: Man as a System Component, and System as Man's Environment. Based on a man's presentation of the state of the art a set...... of design criteria is suggested and their relevance discussed. The point is to focus on the operator rather than on the computer. The crucial question is not to program the computer to work on its own conditions, but to “program” the operator to function on human conditions....
-matter definitions are vital legal tools to determine what is currently regulated in established fields of law and whether there is room for a new legal field – Enhancement Law. This paper provides a reflection on the relevance of establishing a legal definition of human enhancement and to what extent different...... legal fields and jurisdictions may warrant different understandings of such concept. It reviews a number of different and often divergent concepts and taxonomies of human enhancement and concludes with the proposal and analysis of a definition: Use of technological means with the intention to improve......Emerging technologies open the prospect of extraordinary interventions on the human body. These may go beyond what is strictly necessary to sustain health and well-being. While responding to social and ethical challenges of such advances, the Law simultaneously faces the challenge of reflecting...
This document presents human factors guides, which have been developed in order to provide licensees of the AECB with advice as to how to address human factors issues within the design and assessment process. This documents presents the results of a three part study undertaken to develop three guides which are enclosed in this document as Parts B, C and D. As part of the study human factors standards, guidelines, handbooks and other texts were researched, to define those which would be most useful to the users of the guides and for the production of the guides themselves. Detailed specifications were then produced to outline the proposed contents and format of the three guides. (author). 100 refs., 3 tabs., 11 figs
Penington, J [PHF Services Inc., (Canada)
This document presents human factors guides, which have been developed in order to provide licensees of the AECB with advice as to how to address human factors issues within the design and assessment process. This documents presents the results of a three part study undertaken to develop three guides which are enclosed in this document as Parts B, C and D. As part of the study human factors standards, guidelines, handbooks and other texts were researched, to define those which would be most useful to the users of the guides and for the production of the guides themselves. Detailed specifications were then produced to outline the proposed contents and format of the three guides. (author). 100 refs., 3 tabs., 11 figs.
Rochlis-Zumbado, Jennifer; Sandor, Aniko; Ezer, Neta
Risk of Inadequate Design of Human and Automation/Robotic Integration (HARI) is a new Human Research Program (HRP) risk. HRI is a research area that seeks to understand the complex relationship among variables that affect the way humans and robots work together to accomplish goals. The DRP addresses three major HRI study areas that will provide appropriate information for navigation guidance to a teleoperator of a robot system, and contribute to the closure of currently identified HRP gaps: (1) Overlays -- Use of overlays for teleoperation to augment the information available on the video feed (2) Camera views -- Type and arrangement of camera views for better task performance and awareness of surroundings (3) Command modalities -- Development of gesture and voice command vocabularies
Knee, H.E.; Schryver, J.C.
Models of human behavior and cognition (HB and C) are necessary for understanding the total response of complex systems. Many such models have come available over the past thirty years for various applications. Unfortunately, many potential model users remain skeptical about their practicality, acceptability, and usefulness. Such hesitancy stems in part to disbelief in the ability to model complex cognitive processes, and a belief that relevant human behavior can be adequately accounted for through the use of commonsense heuristics. This paper will highlight several models of HB and C and identify existing and potential applications in attempt to dispel such notions. (author)
van Spijk, Piet
If it is true that health is a priority objective of medicine, then medical practice can only be successful if the meaning of the term "health" is known. Various attempts have been made over the years to define health. This paper proposes a new definition. In addition to current health concepts, it also takes into account the distinction between specifically human (great) health and health as the absence of disease and illness-i.e. small health. The feeling of leading a life that makes sense plays a key role in determining specifically human great health.
Ayuo, P O
To provide an overview of risk factors, presentation and management of human cryptosporidium infection. Literature review was obtained through PubMed search. Published articles on the taxonomy of Cryptosporidium and the epidemiology, clinical presentation and management of cryptosporidiosis were reviewed. Abstracts and complete articles relevant to the objective were selected, read and analysed to extract information for this article. Human cryptosporidiosis is a severe diarrhoeal disease in malnourished children and immuno-compromised adults in whom it confers poor prognosis. Management is mainly supportive as drug therapy remains elusive. Fortunately the prevalence in AIDS patients is declining due to the widespread use of combination antiretroviral therapy (cART).
Grier, David Alan
Before Palm Pilots and iPods, PCs and laptops, the term ""computer"" referred to the people who did scientific calculations by hand. These workers were neither calculating geniuses nor idiot savants but knowledgeable people who, in other circumstances, might have become scientists in their own right. When Computers Were Human represents the first in-depth account of this little-known, 200-year epoch in the history of science and technology. Beginning with the story of his own grandmother, who was trained as a human computer, David Alan Grier provides a poignant introduction to the wider wo
Barnett, Ralph L; Liber, Theodore
Use of unassisted human push capability arises from time to time in the areas of crowd and animal control, the security of locked doors, the integrity of railings, the removal of tree stumps and entrenched vehicles, the manoeuvering of furniture, and athletic pursuits such as US football or wrestling. Depending on the scenario, human push capability involves strength, weight, weight distribution, push angle, footwear/floor friction, and the friction between the upper body and the pushed object. Simple models are used to establish the relationships among these factors.
McKenna, Dennis J
In this essay, the author shares his personal reflections gleaned from a lifetime of research with ayahuasca, and speculates on the societal, political, planetary, and evolutionary implications of humanity's aeons-old symbiosis with this shamanic plant. The thesis is developed that at this critical historical juncture, ayahuasca has developed a strategy to broadcast its message to a wider world--a reflection of the urgent need to avert global ecological catastrophe. While ayahuasca has much to teach us, the critical question is, will humanity hear it, and heed it, in time?
This article analyses the United Nations (UN) Guidelines on Business and Human Rights adopted in 2011 by the UN Human Rights Council from the perspective of Transnational Business Governance Interactions (TBGI) analytical framework (Eberlein et al. 2014). The article identifies and discusses...... that the UN Guiding Principles are unique in several respects of relevance to transnational business governance interaction and indicate the relevance of the TBGI approach to public regulatory transnational business governance initiatives. The analysis of the Guiding Principles as interactional transnational...... business governance suggests that this form of governance offers prospects for public institutions as a means towards regulating global sustainability concerns....
Knudsen, A B; Larsen, M; Mackey, Abigail
The myotendinous junction (MTJ) is a specialized structure in the musculotendinous system, where force is transmitted from muscle to tendon. Animal models have shown that the MTJ takes form of tendon finger-like processes merging with muscle tissue. The human MTJ is largely unknown and has never...... been described in three dimensions (3D). The aim of this study was to describe the ultrastructure of the human MTJ and render 3D reconstructions. Fourteen subjects (age 25 ± 3 years) with isolated injury of the anterior cruciate ligament (ACL), scheduled for reconstruction with a semitendinosus...
Mady, Carlos Eduardo Keutenedjian
The exergy analysis of the human body is a tool that can provide indicators of health and life quality. To perform the exergy balance it is necessary to calculate the metabolism on an exergy basis, or metabolic exergy, although there is not yet consensus in its calculation procedure. Hence, the aim of this work is to provide a general method to evaluate this physical quantity for human body based on indirect calorimetry data. To calculate the metabolism on an exergy basis it is necessary to d...
Carlos López Dawson
Full Text Available In the formation of a new Constitution the constituents will require to know or reach an agreement on the nature of human rights; then, to determine how the State will enforce the respect to those rights. To do so, it is necessary to resort to the history and evolution of these rights, and the present work aims to contribute to an efficient productive debate about the nature of human rights, so that citizens can decide on the understanding that this is a thoughtful democratic and humanistic founded decision. The analysis is in the actual technical-ideological republican system which correspond to the current state of international law
to become part of a global cultural flow, thus calling into question the physical connection between viewer and image. This article analyses what happens to that connection when not only the image but also the physical body is mediated and challenged in post-human relations, and examines the ensuing ethical...... implications. The author takes photojournalism and, in particular, mobile phone footage as a starting point for an exploration of the (post-human) body as evidence and sign of authenticity in the modern age of digital communications and journalism....
This article analyses the United Nations (UN) Guidelines on Business and Human Rights adopted in 2011 by the UN Human Rights Council from the perspective of transnational business governance interactions (TBGI) analytical framework.1 The article identifies and discusses dimensions of interaction...... and components of regulatory governance which characterize the Guiding Principles, focusing in particular on rule formation and implementation. The article notes that the Guiding Principles actively enrolled other actors for the rule-making process, ensuring support in a politically and legally volatile field...
This volume addresses the emerging area of human computation, The chapters, written by leading international researchers, explore existing and future opportunities to combine the respective strengths of both humans and machines in order to create powerful problem-solving capabilities. The book bridges scientific communities, capturing and integrating the unique perspective and achievements of each. It coalesces contributions from industry and across related disciplines in order to motivate, define, and anticipate the future of this exciting new frontier in science and cultural evolution. Reade
McClennen, Sophia A.
In her paper "The Humanities, Human Rights, and the Comparative Imagination" Sophia A. McClennen argues that understanding the relationship between culture and human rights depends on humanist perspectives attentive to the relationship between storytelling and identity, mass culture and ideology, text and audience, critical thinking and engaged citizenship. After briefly considering how the divide between the humanities and human rights advocates developed and how it might best be overcome, s...
Stambolian, Damon B.; Lawrence, Brad A.; Stelges, Katrine S.; Steady, Marie-Jeanne O.; Ridgwell, Lora C.; Mills, Robert E.; Henderson, Gena; Tran, Donald; Barth, Tim
There have been many advancements and accomplishments over the last few years using human modeling for human factors engineering analysis for design of spacecraft. The key methods used for this are motion capture and computer generated human models. The focus of this paper is to explain the human modeling currently used at Kennedy Space Center (KSC), and to explain the future plans for human modeling for future spacecraft designs
Full Text Available As a constitutional principle of the human rights, the human dignity is a supreme value, a norm and a right, thus that the reconfiguration of protection standards of fundamental human rights is made by cohesion of the legal, social and moral dimensions of human dignity. With this article, the author argues that legal meaning, social meaning and moral meaning of human dignity, are centerpiece of protection of freedom under law.
Cary, Michael S.
Describes the current impoverishment of the humanities and the gulf separating the humanities from the sciences. Discusses the need for adequate humanities instruction at the elementary-secondary level. Suggests that humanities teachers rediscover the Italian Renaissance spirit to improve their teaching. (SB)
This essay uses the recent victory of an IBM computer (Watson) in the TV game, "Jeopardy," to speculate on the abilities Watson would need, in addition to those it has, to be human. The essay's basic premise is that to be human is to behave as humans behave and to function in society as humans function. Alternatives to this premise are considered…
Devine, Carol; Hansen, Carol Rae; Wilde, Ralph; Bronkhorst, Daan; Moritz, Frederic A.; Rolle, Baptiste; Sherman, Rebecca; Southard, Jo Lynn; Wilkinson, Robert; Poole, Hilary, Ed.
This reference work documents the history of human rights theory, explains each article of the Universal Declaration of Human Rights, explores the contemporary human rights movement, and examines the major human rights issues facing the world today. This book is the first to combine historical and contemporary perspectives on these critical…
Yoshikawa, Hidekazu; Furuta, Kazuo.
Review on progress of research and development on human modeling methods is made from the viewpoint of its importance on total man-machine system reliability surrounding nuclear power plant operation. Basic notions on three different approaches of human modeling (behavioristics, cognitives and sociologistics) are firstly introduced, followed by the explanation of fundamental scheme to understand human cognitives at man-machine interface and the mechanisms of human error and its classification. Then, general methodologies on human cognitive model by AI are explained with the brief summary of various R and D activities now prevailing in the human modeling communities around the world. A new method of dealing with group human reliability is also introduced which is based on sociologistic mathematical model. Lastly, problems on human model validation are discussed, followed by the introduction of new experimental method to estimate human cognitive state by psycho-physiological measurement, which is a new methodology plausible for human model validation. (author)
Charles A. Lewis
Gardening produces not only vegetables and flowers, but also social and behavioral benefits. In low-income housing sites in New York, Philadelphia, and Chicago, gardening programs have resulted in reduced vandalism, new neighborliness, cleaned and painted buildings and streets, and other improvements. The human response to plants, and the qualities of plants that...
Peiris, M; Yen, H-L
Influenza type A viruses affect humans and other animals and cause significant morbidity, mortality and economic impact. Influenza A viruses are well adapted to cross species barriers and evade host immunity. Viruses that cause no clinical signs in wild aquatic birds may adapt in domestic poultry to become highly pathogenic avian influenza viruses which decimate poultry flocks. Viruses that cause asymptomatic infection in poultry (e.g. the recently emerged A/H7N9 virus) may cause severe zoonotic disease and pose a major pandemic threat. Pandemic influenza arises at unpredictable intervals from animal viruses and, in its global spread, outpaces current technologies for making vaccines against such novel viruses. Confronting the threat of influenza in humans and other animals is an excellent example of a task that requires a One Health approach. Changes in travel, trade in livestock and pets, changes in animal husbandry practices, wet markets and complex marketing chains all contribute to an increased risk of the emergence of novel influenza viruses with the ability to cross species barriers, leading to epizootics or pandemics. Coordinated surveillance at the animal- human interface for pandemic preparedness, risk assessment, risk reduction and prevention at source requires coordinated action among practitioners in human and animal health and the environmental sciences. Implementation of One Health in the field can be challenging because of divergent short-term objectives. Successful implementation requires effort, mutual trust, respect and understanding to ensure that long-term goals are achieved without adverse impacts on agricultural production and food security.
Garcia Gutierrez, A.; Gil, C.
The objective of the Program consists in the excellence actuation, achieving the client success with a perfect realisation project. This program consists of different basic elements to reduce the human mistakes: the HuP tools, coaching, learning clocks and iKnow website. There is, too, a document file to consult and practice. All these elements are expounded in this paper.
Sweet, William; Masciulli, Joseph
In this article, the authors review some contemporary cases where biotechnologies have been employed, where they have had global implications, and where there has been considerable debate. The authors argue that the concept of dignity, which lies at the center of such documents as the 2005 Universal Declaration on Bioethics and Human Rights, the…
Amsinck Boie, Hans Nikolaj; Torp, Kristian
adequately be addressed without including the approach to the problem taken in practice; Corporate Social Responsibility, CSR. The book therefore draws upon the concept of CSR and the approaches developed here and discusses whether states may utilize the CSR-based concept of human rights due diligence...
Evolutionary psychology claims to offer a unified perspective on human nature and culture, which can serve to further the integration of psychology and the social sciences. I describe four approaches to evolutionary psychology, and note increasing attention to the agency of the individual in
Lieberman, David A.
This innovative textbook is the first to integrate learning and memory, behaviour, and cognition. It focuses on fascinating human research in both memory and learning (while also bringing in important animal studies) and brings the reader up to date with the latest developments in the subject. Students are encouraged to think critically: key…
Keemink, Arvid Quintijn Leon
This dissertation covers three aspects of upper-extremity exoskeleton design: 1) Kinematics & motion: How to support the full range of motion of the human shoulder? We present a 2D visualization method that can show coupling between the range of motion (ROM) of rotations of the glenohumeral joint.
Jefferson, Andrew M.; Gaborit, Liv Stoltze
Drawing on participatory action research conducted in Sierra Leone, Kosovo and the Philippines, Human Rights in Prisons analyses encounters between rights-based non-governmental organisations and prisons. It explores the previously under-researched perspectives of prison staff and prisoners...
Abstract Following an increase in Roma migration under the European “freedom of movement” laws, Swedish municipalities initiated more than 80 evictions of informal Roma settlements on the grounds of poor sanitation between 2013 and 2016. These evictions echo policies from earlier in the 20th century, when Roma living in Sweden were often marginalized through the denial of access to water and sanitation facilities. The recent Swedish evictions also follow similar government actions across Europe, where Roma settlements are controlled through the denial of access to water and sanitation. However, access to water and sanitation—central aspects of human health—are universal human rights that must be available to all people present in a jurisdiction, regardless of their legal status. The evictions described here violated Sweden’s obligations under both European and international human rights law. More positive government responses are required, such as providing shelters or camping sites, setting up temporary facilities, and directly engaging with communities to address water and sanitation issues. The authors conclude by providing guidance on how states and municipalities can meet their human rights obligations with respect to water and sanitation for vulnerable Roma individuals and informal settlements in their communities. PMID:29302163
Frith, Christopher; Frith, Uta
We review a diversity of studies of human social interaction and highlight the importance of social signals. We also discuss recent findings from social cognitive neuroscience that explore the brain basis of the capacity for processing social signals. These signals enable us to learn about...
Davelaar, E.J.; Raaijmakers, J.G.W.; Hills, T.T.; Robbins, T.W.; Todd, P.M.
The importance of understanding human memory search is hard to exaggerate: we build and live our lives based on what whe remember. This chapter explores the characteristics of memory search, with special emphasis on the use of retrieval cues. We introduce the dependent measures that are obtained
Frank, Eric, Ed.
Describes three human resource development activities: training, education, and development. Explains marketing from the practitioners's viewpoint in terms of customer orientation; external and internal marketing; and market analysis, research, strategy, and mix. Shows how to design, develop, and implement strategic marketing plans and identify…
Hasliza Abdul-Halim; Elaine Ee; T. Ramayah; Noor Hazlina Ahmad
The existing literature on partnership seems to take the relationship between partnership quality and outsourcing success for granted. Therefore, this article aims at examining the role of service quality in strengthening the relationship between partnership quality and human resource (HR) outsourcing success. The samples were obtained from 96 manufacturing organizations in Penang, Malaysia. The results showed that par...
Martinez, Michael E.
The human mind has two types of memory: short-term and long-term. In all types of learning, it is best to use that structure rather than to fight against it. One way to do that is to ensure that learners can fit new information into patterns that can be stored in and more easily retrieved from long-term memory.
Manrique, Pilar; Bolduc, Benjamin; Walk, Seth T.; Oost, van der John; Vos, de Willem M.; Young, Mark J.
The role of bacteriophages in influencing the structure and function of the healthy human gut microbiome is unknown. With few exceptions, previous studies have found a high level of heterogeneity in bacteriophages from healthy individuals. To better estimate and identify the shared phageome of
Rogowitz, B.E.; van Rikxoort, Eva M.; van den Broek, Egon; Pappas, T.N.; Schouten, Theo E.; Daly, S.J.
In an attempt to mimic human (colorful) texture classification by a clustering algorithm three lines of research have been encountered, in which as test set 180 texture images (both their color and gray-scale equivalent) were drawn from the OuTex and VisTex databases. First, a k-means algorithm was
Buur, Jacob; Caglio, Agnese; Jensen, Lars Christian
, a method developed to engage people from different backgrounds in collaboratively analysing videos with the help of physical objects. We will present one of these tools, Action Scrabble, for analysing temporal organisation of human actions. We work with a case of skilled forklift truck driving...
Human health is influenced by pollutants in the air. Since people spend over 80% of their time indoors, indoor air quality may be more related to health problems than outdoor air qual-ity. Indoor air quality is deteriorating because of energy conservation
Capalbo, Antonio; Hoffmann, Eva R.; Cimadomo, Danilo
to chromosome segregation in meiosis and mitosis. OUTCOMES Advances in genomic and imaging technologies are allowing unprecedented insight into chromosome segregation in human oocytes. This includes the identification of a novel chromosome segregation error, termed reverse segregation, as well as sister...
this disease, training of technicians in anti-tuberculosis centers would be the most realistic attitude to detect mycobacteria and/or Paragonimus eggs during the same sputum examination. Key words: Paragonimus spp., Africa, human paragonimiasis, intermediate hosts, tuberculosis. Résumé. Une revue sur la paragonimose ...
Fuel Cells • Artificial Photosynthesis Overview of Topic Areas 3003 Human Performance/Biosystems • Photo-Electro-Magnetic Stimulation of...1) Electronic transport in bacterial nanowires was demonstrated using nanofabrication enabled approaches (2) Identified the biophysical... bacterial nanowires and outer-membrane vesicles enhancing the electron transfer and respiration of individual cells Outlook The first demonstration
Gabrielli, Simona; Totino, Valentina; Macchioni, Fabio; Zuñiga, Freddy; Rojas, Patricia; Lara, Yuni; Roselli, Mimmo; Bartoloni, Alessandro; Cancrini, Gabriella
To investigate human babesiosis in the Bolivian Chaco, in 2013 we tested blood samples from 271 healthy persons living in 2 rural communities in this region. Microscopy and PCR indicated that 3.3% of persons were positive for Babesia microti parasites (US lineage); seroprevalence was 45.7%. Appropriate screening should mitigate the risk for transfusion-associated babesiosis.
Despite high diversity in species as well as metamorphological life-stages, edible insects are essentially an animal-source food contributing high quality protein and fat when viewed in the context of human nutrition. The nutritional contribution of insects to diets in populations where insects ...
Claremont Univ. Center, CA.
This program provides information to students about human sexual biology, behavior and attitudes. The primary intent of the workshops described is to provide fuller information and opportunity for self awareness to encourage participants to be more responsible as sexual beings, and to restructure their attitudes. The program presents the…
Smarr, Melissa M; Sapra, Katherine J; Gemmill, Alison
Fecundity, the biologic capacity to reproduce, is essential for the health of individuals and is, therefore, fundamental for understanding human health at the population level. Given the absence of a population (bio)marker, fecundity is assessed indirectly by various individual-based (e.g. semen ...
Benschop, Kimberley S. M.; Schinkel, Janke; Luken, Manon E.; van den Broek, Peter J. M.; Beersma, Matthias F. C.; Menelik, Negassi; van Eijk, Hetty W. M.; Zaaijer, Hans L.; Vandenbroucke-Grauls, Christina M. J. E.; Beld, Marcel G. H. M.; Wolthers, Katja C.
We identified a novel human parechovirus (HPeV) type (K251176-02) from a neonate with fever. Analysis of the complete genome showed K251176-02 to be a new HPeV genotype. Since K251176-02 could not be neutralized with antibodies against known HPeV serotypes 1-3, it should be classified as a fourth
Lopes, Arminda; Ørngreen, Rikke
This book constitutes the thoroughly refereed post-conference proceedings of the Third IFIP WG 13.6 Working Conference on Human Work Interaction Design, HWID 2012, held in Copenhagen, Denmark, in December 2012. The 16 revised papers presented were carefully selected for inclusion in this volume...
An international conference, Human Genome I, was held Oct. 2-4, 1989 in San Diego, Calif. Selected speakers discussed: Current Status of the Genome Project; Technique Innovations; Interesting regions; Applications; and Organization - Different Views of Current and Future Science and Procedures. Posters, consisting of 119 presentations, were displayed during the sessions. 119 were indexed for inclusion to the Energy Data Base
The Human and Organizational Factors Approach to Industrial Safety (HOFS) consists of identifying and putting in place conditions which encourage a positive contribution from operators (individually and in a team) with regards to industrial safety. The knowledge offered by the HOFS approach makes it possible better to understand what conditions human activity and to act on the design of occupational situations and the organization, in the aim of creating the conditions for safe work. Efforts made in this area can also lead to an improvement in results in terms of the quality of production or occupational safety (incidence and seriousness rates) (Daniellou, F., et al., 2011). Research on industrial accidents shows that they rarely happen as a result of a single event, but rather emerge from the accumulation of several, often seemingly trivial, malfunctions, misunderstandings, incorrect assumptions and other issues. The nuclear community has established rigorous international safety standards and concepts to ensure the protection of people and the environment from harmful effects of ionizing radiation (IAEA, 2014). A review of major human induced disasters in a number of countries and in different industries yields insights into several of the human and organizational factors involved in their occurrence. Some of these factors relate to failures in: • Design or technology; • Training; • Decision making; • Communication; • Preparation for the unexpected; • Understanding of organizational interdependencies
Steven W Cole
Full Text Available A growing literature in human social genomics has begun to analyze how everyday life circumstances influence human gene expression. Social-environmental conditions such as urbanity, low socioeconomic status, social isolation, social threat, and low or unstable social status have been found to associate with differential expression of hundreds of gene transcripts in leukocytes and diseased tissues such as metastatic cancers. In leukocytes, diverse types of social adversity evoke a common conserved transcriptional response to adversity (CTRA characterized by increased expression of proinflammatory genes and decreased expression of genes involved in innate antiviral responses and antibody synthesis. Mechanistic analyses have mapped the neural "social signal transduction" pathways that stimulate CTRA gene expression in response to social threat and may contribute to social gradients in health. Research has also begun to analyze the functional genomics of optimal health and thriving. Two emerging opportunities now stand to revolutionize our understanding of the everyday life of the human genome: network genomics analyses examining how systems-level capabilities emerge from groups of individual socially sensitive genomes and near-real-time transcriptional biofeedback to empirically optimize individual well-being in the context of the unique genetic, geographic, historical, developmental, and social contexts that jointly shape the transcriptional realization of our innate human genomic potential for thriving.
Ubiquitous computing means network connectivity everywhere, linking devices and systems as small as a drawing pin and as large as a worldwide product distribution chain. What could happen when people are so readily networked? This paper explores issues arising from two possible emerging models of ubiquitous human computing: fungible networked brainpower and collective personal vital sign monitoring.
Full Text Available Introduction: Selenium is an essential element for human health and it is toxic at high concentrations. Selenium is a constituent component of selenoproteins that have enzymatic and structural roles in human biochemistry. Selenium is a best antioxidant and catalyst for production of thyroid hormone. This element has the key role in the immune function; prevention of AIDS progression and the deactivity of toxins. Furthermore, selenium is essential for sperm motility and can reduce abortions. Selenium deficiency was also associated with adverse mood states. The findings regarding cardiovascular disease risk related to selenium deficiency is unclear, though other conditions such as vascular inflammation, oxidative stress and selenium deficiency can cause this disease too. Moreover, consuming of 60 mg of selenium per day may be associated with reduction of cancer risk. In this study, a review of studies has been performed on the biochemical function of selenium toxicity, and its effects on human health. Furthermore, certain identified cancers associated with selenium have been discussed to absorb more attention to the status of this element and also as a guide for further studies. Selenium plays the dual character (useful and harmful in human health, and then it is necessary to determine the concentration of this element in body fluids and tissues. An appropriate method for routine measurement of selenium in clinical laboratories is electro thermal atomic absorption spectrometry (ETAAS with very low detection limit and good precision.
Lebret, E [RIVM-National Inst. of Public Health and Environmental Protection (Netherlands)
This article describes some of the features of the assessment of human exposure to environmental pollutants in epidemiological studies. Since exposure assessment in air pollution epidemiology studies typically involve professionals from various backgrounds, interpretation of a concepts like `exposure` may vary. A brief descriptions is therefore given by way of introduction
Gonçalves, Frederica; Campos, Pedro; Clemmensen, Torkil
In this paper, we review research in the emerging practice and research field of Human Work Interaction Design (HWID). We present a HWID frame-work, and a sample of 54 papers from workshops, conferences and journals from the period 2009-2014. We group the papers into six topical groups, and then ...
Lebret, E. [RIVM-National Inst. of Public Health and Environmental Protection (Netherlands)
This article describes some of the features of the assessment of human exposure to environmental pollutants in epidemiological studies. Since exposure assessment in air pollution epidemiology studies typically involve professionals from various backgrounds, interpretation of a concepts like `exposure` may vary. A brief descriptions is therefore given by way of introduction
, men også arbejdssociologien, arbejdspsykologien og human resource development. Den første retning udsprang af de såkaldte Hawthorne-eksperimenter og psykologen Elton Mayos bearbejdelse af resultaterne derfra. Den anden er en løsere gruppering bestående af navne som Abraham Maslow og Frederick Herzberg...
Comstock, George; And Others
To compile a comprehensive review of English language scientific literature regarding the effects of television on human behavior, the authors of this book evaluated more than 2,500 books, articles, reports, and other documents. Rather than taking a traditional approach, the authors followed a new model for the retrieval and synthesis of…
Barnes, M.; Cosenzo, K.; Galster, s.; Hollnagel, E.; Miller, C.; Parasuraman, R.; Reising, J.; Taylor, R.; Breda, L. van
Many versions of future concept of operations (CONOPS) rely heavily on UMVs. The pressure to take the human out of immediate control of these vehicles is being driven by several factors. These factors include a reduction in cost for the production and maintenance of the vehicle, operational
Sandor, Aniko; Cross, E. Vincent, II; Chang, Mai Lee
Human-robot interaction (HRI) is a discipline investigating the factors affecting the interactions between humans and robots. It is important to evaluate how the design of interfaces affect the human's ability to perform tasks effectively and efficiently when working with a robot. By understanding the effects of interface design on human performance, workload, and situation awareness, interfaces can be developed to appropriately support the human in performing tasks with minimal errors and with appropriate interaction time and effort. Thus, the results of research on human-robot interfaces have direct implications for the design of robotic systems. For efficient and effective remote navigation of a rover, a human operator needs to be aware of the robot's environment. However, during teleoperation, operators may get information about the environment only through a robot's front-mounted camera causing a keyhole effect. The keyhole effect reduces situation awareness which may manifest in navigation issues such as higher number of collisions, missing critical aspects of the environment, or reduced speed. One way to compensate for the keyhole effect and the ambiguities operators experience when they teleoperate a robot is adding multiple cameras and including the robot chassis in the camera view. Augmented reality, such as overlays, can also enhance the way a person sees objects in the environment or in camera views by making them more visible. Scenes can be augmented with integrated telemetry, procedures, or map information. Furthermore, the addition of an exocentric (i.e., third-person) field of view from a camera placed in the robot's environment may provide operators with the additional information needed to gain spatial awareness of the robot. Two research studies investigated possible mitigation approaches to address the keyhole effect: 1) combining the inclusion of the robot chassis in the camera view with augmented reality overlays, and 2) modifying the camera
The philosophical foundations of the theory and practice of the creation of cosmic humanity as a process of the evolution of human civilization, the emergence into space, with the prospect of resettlement outside the Earth are considered. There is a connection between myths, fantasies, ideas, concepts and projects aimed at the exploration of outer space, the creation of cosmic humanity. A new and voluminous definition of cosmic humanity in the evolutionary paradigm is given. Cosmic humanity i...
Pragmatism offers a platform for posing relevant questions. This article uses a pragmatic point of departure to question a natural law conception of human rights and to take a closer look at three pressing human rights problems: The human rights situation in states with little or no state capacity......; the revision and adaptation of human rights law; and the not straightforward relationship betweemn human rights and democracy....