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Sample records for counterselective gene replacement

  1. New range of vectors with a stringent 5-fluoroorotic acid-based counterselection system for generating mutants by allelic replacement in Staphylococcus aureus.

    PubMed

    Redder, Peter; Linder, Patrick

    2012-06-01

    We have developed a range of vectors for allelic replacements in Staphylococcus aureus to facilitate genetic work in this opportunistic pathogen. The central feature of the vector range is a selection/counterselection system that takes advantage of the 5-fluoroorotic acid (FOA) resistance and pyrimidine prototrophy caused by the loss and gain, respectively, of the pyrF and pyrE genes. This system allows for stringent counterselection of the vectors during the second homologous recombination of a classic allelic replacement. The basic vector pRLY2, which contains the pyrFE genes from Bacillus subtilis, was combined with chloramphenicol, erythromycin, and tetracycline resistance genes and four different versions of nonreplicative or conditionally replicative origins of replication. The choice between these 12 different pRLY vectors allows for high versatility and ensures that the vectors can be used in virtually any genetic background. Finally, as proof of concept, we present six deletions or modifications of components in the S. aureus degradosome as well as the operon containing the cshB DEAD box helicase.

  2. Counterselection method based on conditional silencing of antitoxin genes in Escherichia coli.

    PubMed

    Tsukuda, Miyuki; Nakashima, Nobutaka; Miyazaki, Kentaro

    2015-11-01

    Counterselection is a genetic engineering technique to eliminate specific genetic fragments containing selectable marker genes. Although the technique is widely used in bacterial genome engineering and plasmid curing experiments, the repertoire of the markers usable in Escherichia coli is limited. Here we developed a novel counterselection method in E. coli based on antisense RNA (asRNA) technology directed against toxin-antitoxin (TA) modules. Under normal conditions, excess antitoxin neutralizes its cognate toxin and thus the module is stably maintained in the genome. We hypothesised that repression of an antitoxin gene would perturb cell growth due to the toxin being released. We designed asRNAs corresponding to all 19 type II antitoxins encoded in the E. coli genome. asRNAs were then conditionally expressed; repression of MqsA in the MqsR/MqsA module had the greatest inhibitory effect, followed by RnlB in the RnlA/RnlB module. The utility of asRNA(MqsA) as a counterselection marker was demonstrated by efficient plasmid curing and strain improvement experiments.

  3. Construction of a Vibrio splendidus mutant lacking the metalloprotease gene vsm by use of a novel counterselectable suicide vector.

    PubMed

    Le Roux, Frédérique; Binesse, Johan; Saulnier, Denis; Mazel, Didier

    2007-02-01

    Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the PBAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.

  4. Construction of a Vibrio splendidus Mutant Lacking the Metalloprotease Gene vsm by Use of a Novel Counterselectable Suicide Vector▿ †

    PubMed Central

    Le Roux, Frédérique; Binesse, Johan; Saulnier, Denis; Mazel, Didier

    2007-01-01

    Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the PBAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected. PMID:17122399

  5. Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker

    PubMed Central

    Jeong, Jaehwan; Seo, Han Na; Jung, Yu Kyung; Lee, Jeewon; Ryu, Gyuri; Lee, Wookjae; Kwon, Euijin; Ryoo, Keunsoo; Kim, Jungyeon; Cho, Hwa-Young; Cho, Kwang Myung; Park, Jin Hwan; Bang, Duhee

    2015-01-01

    Genome engineering can be used to produce bacterial strains with a wide range of desired phenotypes. However, the incorporation of gene-sized DNA fragments is often challenging due to the intricacy of the procedure, off-target effects, and low insertion efficiency. Here we report a genome engineering method enabling the continuous incorporation of gene-sized double-stranded DNAs (dsDNAs) into the Escherichia coli genome. DNA substrates are inserted without introducing additional marker genes, by synchronously turning an endogenous counter-selectable marker gene ON and OFF. To accomplish this, we utilized λ Red protein-mediated recombination to insert dsDNAs within the promoter region of a counter-selectable marker gene, tolC. By repeatedly switching the marker gene ON and OFF, a number of desired gene-sized dsDNAs can be inserted consecutively. With this method, we successfully inserted approximately 13 kb gene clusters to generate engineered E. coli strains producing 1,4-butanediol (1,4-BDO). PMID:25736821

  6. Counter-selection recombineering of the baculovirus genome: a strategy for seamless modification of repeat-containing BACs.

    PubMed

    Westenberg, Marcel; Soedling, Helen M; Mann, Derek A; Nicholson, Linda J; Dolphin, Colin T

    2010-09-01

    Recombineering is employed to modify large DNA clones such as fosmids, BACs and PACs. Subtle and seamless modifications can be achieved using counter-selection strategies in which a donor cassette carrying both positive and negative markers inserted in the target clone is replaced by the desired sequence change. We are applying counter-selection recombineering to modify bacmid bMON14272, a recombinant baculoviral genome, as we wish to engineer the virus into a therapeutically useful gene delivery vector with cell targeting characteristics. Initial attempts to replace gp64 with Fusion (F) genes from other baculoviruses resulted in many rearranged clones in which the counter-selection cassette had been deleted. Bacmid bMON14272 contains nine highly homologous regions (hrs) and deletions were mapped to recombination between hr pairs. Recombineering modifications were attempted to decrease intramolecular recombination and/or increase recombineering efficiency. Of these only the use of longer homology arms on the donor molecule proved effective permitting seamless modification. bMON14272, because of the presence of the hr sequences, can be considered equivalent to a highly repetitive BAC and, as such, the optimized method detailed here should prove useful to others applying counter-selection recombineering to modify BACs or PACs containing similar regions of significant repeating homologies.

  7. Absence of a gene encoding cytosine deaminase in the genome of the agaricomycete Coprinopsis cinerea enables simple marker recycling through 5-fluorocytosine counterselection.

    PubMed

    Nakazawa, Takehito; Honda, Yoichi

    2015-08-01

    Coprinopsis cinerea is a model species for molecular genetics studies of sexual development in agaricomycetes or homobasidiomycetes. Recently, efficient gene targeting was established in this fungus by generating Cc.ku70 or Cc.lig4 disruptants. To determine the molecular mechanisms underlying sexual development, which involves many genes, generating multiple gene disruptants is required. However, the number of transformation markers available for C. cinerea is limited. This problem would be solved by establishing marker recycling. In this study, we found that C. cinerea lacks a gene encoding a homolog of Saccharomyces cerevisiae cytosine deaminase (Fcy1p) in its genome, which is present in many other fungi. We also observed that C. cinerea is resistant to 5-fluorocytosine. Based on these findings, we established a simple marker recycling method in this fungus using 5-fluorocytosine counterselection after heterologous expression of FCY1 derived from Pleurotus ostreatus, together with the hygromycin resistance gene. This study proposes a simple genetic manipulation system that can be performed using wild-type strains of several fungi that lack a gene homologous to S. cerevisiae FCY1 in their genomes.

  8. Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker

    PubMed Central

    Carr, Jennifer F.; Danziger, Michael E.; Huang, Athena L.; Dahlberg, Albert E.

    2015-01-01

    ABSTRACT Thermus thermophilus is an extremely thermophilic bacterium that is widely used as a model thermophile, in large part due to its amenability to genetic manipulation. Here we describe a system for the introduction of genomic point mutations or deletions using a counterselectable marker consisting of a conditionally lethal mutant allele of pheS encoding the phenylalanyl-tRNA synthetase α-subunit. Mutant PheS with an A294G amino acid substitution renders cells sensitive to the phenylalanine analog p-chlorophenylalanine. Insertion of the mutant pheS allele via a linked kanamycin resistance gene into a chromosomal locus provides a gene replacement intermediate that can be removed by homologous recombination using p-chlorophenylalanine as a counterselective agent. This selection is suitable for the sequential introduction of multiple mutations to produce a final strain unmarked by an antibiotic resistance gene. We demonstrated the utility of this method by constructing strains bearing either a point mutation in or a precise deletion of the rrsB gene encoding 16S rRNA. We also used this selection to identify spontaneous, large-scale deletions in the pTT27 megaplasmid, apparently mediated by either of the T. thermophilus insertion elements ISTth7 and ISTth8. One such deletion removed 121 kb, including 118 genes, or over half of pTT27, including multiple sugar hydrolase genes, and facilitated the development of a plasmid-encoded reporter system based on β-galactosidase. The ability to introduce mutations ranging from single base substitutions to large-scale deletions provides a potentially powerful tool for engineering the genome of T. thermophilus and possibly other thermophiles as well. IMPORTANCE Thermus thermophilus is an extreme thermophile that has played an important part in the development of both biotechnology and basic biological research. Its suitability as a genetic model system is established by its natural competence for transformation, but the

  9. Improved seamless mutagenesis by recombineering using ccdB for counterselection

    PubMed Central

    Wang, Hailong; Bian, Xiaoying; Xia, Liqiu; Ding, Xuezhi; Müller, Rolf; Zhang, Youming; Fu, Jun; Stewart, A. Francis

    2014-01-01

    Recombineering, which is the use of homologous recombination for DNA engineering in Escherichia coli, usually uses antibiotic selection to identify the intended recombinant. When combined in a second step with counterselection using a small molecule toxin, seamless products can be obtained. Here, we report the advantages of a genetic strategy using CcdB as the counterselectable agent. Expression of CcdB is toxic to E. coli in the absence of the CcdA antidote so counterselection is initiated by the removal of CcdA expression. CcdB counterselection is robust and does not require titrations or experiment-to-experiment optimization. Because counterselection strategies necessarily differ according to the copy number of the target, we describe two variations. For multi-copy targets, we use two E. coli hosts so that counterselection is exerted by the transformation step that is needed to separate the recombined and unrecombined plasmids. For single copy targets, we put the ccdA gene onto the temperature-sensitive pSC101 Red expression plasmid so that counterselection is exerted by the standard temperature shift to remove the expression plasmid. To reduce unwanted intramolecular recombination, we also combined CcdB counterselection with Redα omission. These options improve the use of counterselection in recombineering with BACs, plasmids and the E. coli chromosome. PMID:24369425

  10. Liquid-Based Iterative Recombineering Method Tolerant to Counter-Selection Escapes

    PubMed Central

    Tominaga, Masahiro; Kawai-Noma, Shigeko; Kawagishi, Ikuro; Sowa, Yoshiyuki; Saito, Kyoichi; Umeno, Daisuke

    2015-01-01

    Selection-based recombineering is a flexible and proven technology to precisely modify bacterial genomes at single base resolution. It consists of two steps of homologous recombination followed by selection/counter-selection. However, the shortage of efficient counter-selectable markers limits the throughput of this method. Additionally, the emergence of ‘selection escapees’ can affect recombinant pools generated through this method, and they must be manually removed at each step of selection-based recombineering. Here, we report a series of efforts to improve the throughput and robustness of selection-based recombineering and to achieve seamless and automatable genome engineering. Using the nucleoside kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the non-natural nucleoside dP, a highly efficient, rapid, and liquid-based counter-selection system was established. By duplicating hsvtk gene, combined with careful control of the population size for the subsequent round, we effectively eliminated selection escapes, enabling seamless and multiple insertions/replacement of gene-size fragments in the chromosome. Four rounds of recombineering could thus be completed in 10 days, requiring only liquid handling and without any need for colony isolation or genotype confirmation. The simplicity and robustness of our method make it broadly accessible for multi-locus chromosomal modifications. PMID:25775434

  11. Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans▿

    PubMed Central

    Xie, Zhoujie; Okinaga, Toshinori; Qi, Fengxia; Zhang, Zhijun; Merritt, Justin

    2011-01-01

    Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed that utilize either temperature-sensitive plasmids or counterselectable suicide vectors containing both positive- and negative-selection markers. For many species, these techniques are not especially useful due to difficulties of cloning with Escherichia coli and/or a lack of functional negative-selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable to a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows extremely quick assembly and eliminates the requirement for either temperature-sensitive replicons or suicide vectors. As a proof of principle, we used Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild-type S. mutans strains, we further demonstrated that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches. PMID:21948849

  12. Gene replacement in Lactobacillus helveticus.

    PubMed Central

    Bhowmik, T; Fernández, L; Steele, J L

    1993-01-01

    An efficient method for gene replacement in Lactobacillus helveticus CNRZ32 was developed by utilizing pSA3 as an integration vector. This plasmid is stably maintained in CNRZ32 at 37 degrees C but is unstable at 45 degrees C. This method consisted of a two-step gene-targeting technique: (i) chromosomal integration of a plasmid carrying an internal deletion in the gene of interest via homologous recombination and (ii) excision of the vector and the wild-type gene via homologous recombination, resulting in gene replacement. By using this procedure, the chromosomal X-prolyl dipeptidyl aminopeptidase gene (pepXP) of CNRZ32 was successfully inactivated. Images PMID:8104928

  13. An upp-based markerless gene replacement method for genome reduction and metabolic pathway engineering in Pseudomonas mendocina NK-01 and Pseudomonas putida KT2440.

    PubMed

    Wang, Yuanyuan; Zhang, Chi; Gong, Ting; Zuo, Zhenqiang; Zhao, Fengjie; Fan, Xu; Yang, Chao; Song, Cunjiang

    2015-06-01

    A markerless gene replacement method was adapted by combining a suicide plasmid, pEX18Tc, with a counterselectable marker, the upp gene encoding uracil phosphoribosyltransferase (UPRTase), for the medium-chain length polyhydroxyalkanoates (PHA(MCL))-producing strain Pseudomonas mendocina NK-01. An NK-01 5-fluorouracil (5-FU) resistant background strain was first constructed by deleting the chromosomal upp gene. The suicide plasmid pEX18Tc, carrying a functional allele of the upp gene of P. mendocina NK-01, was used to construct the vectors to delete the algA (encoding mannose-1-phosphate guanylyltransferase) and phaZ (encoding PHA(MCL) depolymerase) genes, and a 30 kb chromosomal fragment in the 5-FU resistant background host. The genes were removed efficiently from the genome of P. mendocina NK-01 and left a markerless chromosomal mutant. In addition, two exogenous genes were inserted into the phaC1 (PHA(MCL) polymerase) loci of Pseudomonas putida KT-∆UPP simultaneously. Thus, we constructed a genetically stable and marker-free P. putida KT2440 mutant with integrated mpd (encoding methyl parathion hydrolase (MPH)) and pytH (encoding a pyrethroid-hydrolyzing carboxylesterase (PytH)) gene on the chromosome. The upp-based counterselection system could be further adapted for P. mendocina NK-01 and P. putida KT2440 and used for genome reduction and metabolic pathway engineering.

  14. The apt/6-Methylpurine Counterselection System and Its Applications in Genetic Studies of the Hyperthermophilic Archaeon Sulfolobus islandicus

    PubMed Central

    Bi, Hongkai; Whitaker, Rachel J.

    2016-01-01

    ABSTRACT Sulfolobus islandicus serves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker in S. islandicus to expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) in S. islandicus M.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded by M164_0158 (apt). The application of the apt gene as a novel counterselectable marker was first illustrated by constructing an unmarked α-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations in S. islandicus M.16.4 at the apt locus. Moreover, the general conservation of apt genes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that the apt locus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeon S. islandicus. IMPORTANCE Currently, the pyrEF/5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since most Sulfolobus mutants constructed by the research community were derived from genetic hosts lacking the pyrEF genes, the pyrEF/5-FOA system is no longer available for use in forward mutation assays. Demonstration of the apt/6-MP counterselection system for the Sulfolobus model renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with a pyrEF-deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies among S

  15. Counterselection employing mutated pheS for markerless genetic deletion in Bacteroides species.

    PubMed

    Kino, Yasuhiro; Nakayama-Imaohji, Haruyuki; Fujita, Masashi; Tada, Ayano; Yoneda, Saori; Murakami, Kazuya; Hashimoto, Masahito; Hayashi, Tetsuya; Okazaki, Katsuichiro; Kuwahara, Tomomi

    2016-12-01

    Markerless gene deletion is necessary for multiple gene disruptions due to the limited number of antibiotic resistant markers for some bacteria. However, even in transformable strains, obtaining the expected mutation without a marker requires laborious screening of a large number of colonies. Previous studies had success in various bacteria with a counter-selection system where a conditional lethal gene was incorporated into the vector. We examined the efficacy of the mutated pheS gene (pheS*) as a counter-selective marker for gene deletion in Bacteroides. This mutation produces an amino acid substitution (A303G) in the alpha subunit of Bacteroides phenylalanyl tRNA synthetase, which in E. coli alters the specificity of the tRNA synthetase resulting in a conditional lethal mutation due to the incorporation of p-chloro-phenylalanine (p-Cl-Phe) into protein. B. fragilis YCH46 and B. thetaiotaomicron VPI-5482 transformed with a pheS*-harboring shuttle vector were clearly growth-inhibited in the presence of >5 mM p-Cl-Phe in liquid defined minimal media (DMM) and on DMM agar plates. A targeting plasmid was constructed to delete the genetic region for capsular polysaccharide PS2 in B. fragilis or PS1 in B. thetaiotaomicron. After counterselection, p-Cl-Phe-resistant colonies were generated at a frequency of 8.1 × 10(-3) for B. fragilis and 1.7 × 10(-3) for B. thetaiotaomicron. Of the p-Cl-Phe-resistant colonies, 4.2% and 72% harbored the correct genetic deletion for B. fragilis and B. thetaiotaomicron, respectively. These results indicate that mutated pheS is a useful counter-selective gene to construct markerless genetic deletions in Bacteroides. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Toxicity of indoxyl derivative accumulation in bacteria and its use as a new counterselection principle.

    PubMed

    Angelov, Angel; Li, Haijuan; Geissler, Andreas; Leis, Benedikt; Liebl, Wolfgang

    2013-12-01

    In this work we describe the conditional toxic effect of the expression of enzymes that cleave 5-bromo-4-chloro-3-indolyl (BCI) substrates and its use as a new counterselection principle useful for the generation of clean and unmarked mutations in the genomes of bacteria. The application of this principle was demonstrated in the thermophile Thermus thermophilus HB27 and in a mesophile for which currently no counterselection markers are available, Micrococcus luteus ATCC 27141. For T. thermophilus, the indigogenic substrate BCI-β-glucoside was used in combination with the T. thermophilus β-glucosidase gene (bgl). For M. luteus, a combination of BCI-β-galactoside and the E. coli lacZ gene was implemented. We observed a strong growth-inhibiting effect when the strains were grown on agar plates containing the appropriate BCI substrates, the inhibition being proportional to the substrate concentration and the level of bgl/lacZ expression. The growth inhibition apparently depends on intracellular BCI substrate cleavage and accumulation of toxic indoxyl precipitates. The bgl and lacZ genes were used as counterselection markers for the rapid generation of scar-less chromosomal deletions in T. thermophilus HB27 (both in a Δbgl and in a wild type background) and in M. luteus ATCC 27141. In addition to Thermus and Micrococcus, sensitivity to BCI substrate cleavage was observed for other Gram-negative and Gram-positive species belonging to various bacterial phyla, including representatives of the genera Staphylococcus, Bacillus, Corynebacterium, Rhodococcus, Paracoccus and Xanthomonas. Thus, the toxicity of indoxyl derivative accumulation upon BCI substrate cleavage can be used for selection purposes in a broad range of microorganisms. Copyright © 2013 Elsevier GmbH. All rights reserved.

  17. Development of mitochondrial gene replacement therapy.

    PubMed

    Khan, Shaharyar M; Bennett, James P

    2004-08-01

    Many "classic" mitochondrial diseases have been described that arise from single homoplasmic mutations in mitochondrial DNA (mtDNA). These diseases typically affect nonmitotic tissues (brain, retina, muscle), present with variable phenotypes, can appear sporadically, and are untreatable. Evolving evidence implicates mtDNA abnormalities in diseases such as Alzheimer's, Parkinson's, and type II diabetes, but specific causal mutations for these conditions remain to be defined. Understanding the mtDNA genotype-phenotype relationships and developing specific treatment for mtDNA-based diseases is hampered by inability to manipulate the mitochondrial genome. We present a novel protein transduction technology ("protofection") that allows insertion and expression of the human mitochondrial genome into mitochondria of living cells. With protofection, the mitochondrial genotype can be altered, or exogenous genes can be introduced to be expressed and either retained in mitochondria or be directed to other organelles. Protofection also delivers mtDNA in vivo, opening the way to rational development of mitochondrial gene replacement therapy of mtDNA-based diseases.

  18. Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker

    PubMed Central

    Heap, John T.; Ehsaan, Muhammad; Cooksley, Clare M.; Ng, Yen-Kuan; Cartman, Stephen T.; Winzer, Klaus; Minton, Nigel P.

    2012-01-01

    Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms. PMID:22259038

  19. Double targeted gene replacement for creating null mutants.

    PubMed Central

    Cruz, A; Coburn, C M; Beverley, S M

    1991-01-01

    We have used double gene targeting to create homozygous gene replacements in the protozoan parasite Leishmania major, an asexual diploid. This method uses two independent selectable markers in successive rounds of gene targeting to replace both alleles of an endogenous gene. We developed an improved hygromycin B-resistance cassette encoding hygromycin phosphotransferase (HYG) for use as a selectable marker for Leishmania. HYG-containing vectors functioned equivalently to those containing the neomycin phosphotransferase (NEO) cassette previously used for extrachromosomal transformation or gene targeting. Drug resistances conferred by the NEO and HYG markers were independent, allowing simultaneous selection for both markers. A HYG targeting vector was utilized to replace the single dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene remaining in a line heterozygous for a NEO replacement at the dhfr-ts locus (+/neo), with a targeting efficiency comparable to that seen with wild-type recipients. The resultant dhfr-ts- line (hyg/neo) was auxotrophic for thymidine. The double targeted replacement method will enable functional genetic testing in a variety of asexual diploids, including cultured mammalian cells and fungi such as Candida albicans. Additionally, it may be possible to use Leishmania bearing conditionally auxotrophic gene replacements as safe, improved live vaccines for leishmaniasis. Images PMID:1651496

  20. Double targeted gene replacement for creating null mutants.

    PubMed

    Cruz, A; Coburn, C M; Beverley, S M

    1991-08-15

    We have used double gene targeting to create homozygous gene replacements in the protozoan parasite Leishmania major, an asexual diploid. This method uses two independent selectable markers in successive rounds of gene targeting to replace both alleles of an endogenous gene. We developed an improved hygromycin B-resistance cassette encoding hygromycin phosphotransferase (HYG) for use as a selectable marker for Leishmania. HYG-containing vectors functioned equivalently to those containing the neomycin phosphotransferase (NEO) cassette previously used for extrachromosomal transformation or gene targeting. Drug resistances conferred by the NEO and HYG markers were independent, allowing simultaneous selection for both markers. A HYG targeting vector was utilized to replace the single dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene remaining in a line heterozygous for a NEO replacement at the dhfr-ts locus (+/neo), with a targeting efficiency comparable to that seen with wild-type recipients. The resultant dhfr-ts- line (hyg/neo) was auxotrophic for thymidine. The double targeted replacement method will enable functional genetic testing in a variety of asexual diploids, including cultured mammalian cells and fungi such as Candida albicans. Additionally, it may be possible to use Leishmania bearing conditionally auxotrophic gene replacements as safe, improved live vaccines for leishmaniasis.

  1. Gene replacement therapy for hereditary emphysema

    SciTech Connect

    Skolnick, A.

    1989-11-10

    Investigators suggest that human trials of gene therapy to correct a genetic disorder that usually leads to emphysema early in life may be only a few years away. Speaking at the American Lung Association's Second Annual Science Writers' Forum, R. G. Crystal, chief of the Pulmonary Branch of the National Heart, Lung, and Blood Institute offered an explanation of how hereditary emphysema may be more amenable to genetic therapy than other such diseases. In persons who lack a functioning gene for alpha{sup 1}-antitrypsin, a proteolytic enzyme, neutrophil elastase, attacks the walls of the lungs' alveoli, eventually leading to progressive pulmonary function loss. Two animal models of gene insertion are described.

  2. A method for highly efficient exact gene replacement without selection

    PubMed Central

    Cross, Frederick R.; Pecani, Kresti

    2013-01-01

    We describe a highly efficient method for exact gene replacement in budding yeast. Induction of rapid and efficient recombination in an entire cell population results in at least 50% of the recombinants undergoing a specific switch of the endogenous copy to a specific mutated allele, with no remaining markers or remnant of foreign DNA, without selection. To accomplish this, a partial copy of the replacement allele, followed by an HO cut site, is installed adjacent to the wild-type locus, in a GAL-HO MATa-inc background. HO induction results in near-quantitative site cleavage and recombination/gene conversion, resulting in either regeneration of wild-type, or switch of the endogenous allele to the mutant, with accompanying deletion of intervening marker sequences, yielding an exact replacement. Eliminating the need for selection (over days) of rare recombinants removes concerns about second-site suppressor mutations, and also allows direct phenotypic analysis even of lethal gene replacements without the need of a method to make the lethality conditional, or to employ regulated promoters of unknown strength compared to the endogenous promoter. To test this method, we tried two known lethal gene replacements, substituting the non-essential CDH1 gene with a dominantly lethal version mutated for its Cdk phosphorylation sites, and substituting the essential CDC28 gene with two recessively lethal versions, one containing an early stop codon and another one inactivating Cdc28 kinase activity. We also tested a gene replacement of unknown phenotypic consequences: replacing the non-essential CLB3 B-type cyclin with a version lacking its destruction box. PMID:21246629

  3. Replacing and Additive Horizontal Gene Transfer in Streptococcus

    PubMed Central

    Choi, Sang Chul; Rasmussen, Matthew D.; Hubisz, Melissa J.; Gronau, Ilan; Stanhope, Michael J.; Siepel, Adam

    2012-01-01

    The prominent role of Horizontal Gene Transfer (HGT) in the evolution of bacteria is now well documented, but few studies have differentiated between evolutionary events that predominantly cause genes in one lineage to be replaced by homologs from another lineage (“replacing HGT”) and events that result in the addition of substantial new genomic material (“additive HGT”). Here in, we make use of the distinct phylogenetic signatures of replacing and additive HGTs in a genome-wide study of the important human pathogen Streptococcus pyogenes (SPY) and its close relatives S. dysgalactiae subspecies equisimilis (SDE) and S. dysgalactiae subspecies dysgalactiae (SDD). Using recently developed statistical models and computational methods, we find evidence for abundant gene flow of both kinds within each of the SPY and SDE clades and of reduced levels of exchange between SPY and SDD. In addition, our analysis strongly supports a pronounced asymmetry in SPY–SDE gene flow, favoring the SPY-to-SDE direction. This finding is of particular interest in light of the recent increase in virulence of pathogenic SDE. We find much stronger evidence for SPY–SDE gene flow among replacing than among additive transfers, suggesting a primary influence from homologous recombination between co-occurring SPY and SDE cells in human hosts. Putative virulence genes are correlated with transfer events, but this correlation is found to be driven by additive, not replacing, HGTs. The genes affected by additive HGTs are enriched for functions having to do with transposition, recombination, and DNA integration, consistent with previous findings, whereas replacing HGTs seen to influence a more diverse set of genes. Additive transfers are also found to be associated with evidence of positive selection. These findings shed new light on the manner in which HGT has shaped pathogenic bacterial genomes. PMID:22617954

  4. Gene replacement therapy for genetic hepatocellular jaundice.

    PubMed

    van Dijk, Remco; Beuers, Ulrich; Bosma, Piter J

    2015-06-01

    Jaundice results from the systemic accumulation of bilirubin, the final product of the catabolism of haem. Inherited liver disorders of bilirubin metabolism and transport can result in reduced hepatic uptake, conjugation or biliary secretion of bilirubin. In patients with Rotor syndrome, bilirubin (re)uptake is impaired due to the deficiency of two basolateral/sinusoidal hepatocellular membrane proteins, organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3. Dubin-Johnson syndrome is caused by a defect in the ATP-dependent canalicular transporter, multidrug resistance-associated protein 2 (MRP2), which mediates the export of conjugated bilirubin into bile. Both disorders are benign and not progressive and are characterised by elevated serum levels of mainly conjugated bilirubin. Uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) is responsible for the glucuronidation of bilirubin; deficiency of this enzyme results in unconjugated hyperbilirubinaemia. Gilbert syndrome is the mild and benign form of inherited unconjugated hyperbilirubinaemia and is mostly caused by reduced promoter activity of the UGT1A1 gene. Crigler-Najjar syndrome is the severe inherited form of unconjugated hyperbilirubinaemia due to mutations in the UGT1A1 gene, which can cause kernicterus early in life and can be even lethal when left untreated. Due to major disadvantages of the current standard treatments for Crigler-Najjar syndrome, phototherapy and liver transplantation, new effective therapeutic strategies are under development. Here, we review the clinical features, pathophysiology and genetic background of these inherited disorders of bilirubin metabolism and transport. We also discuss the upcoming treatment option of viral gene therapy for genetic disorders such as Crigler-Najjar syndrome and the possible immunological consequences of this therapy.

  5. Gene replacement therapy for retinal CNG channelopathies.

    PubMed

    Schön, Christian; Biel, Martin; Michalakis, Stylianos

    2013-10-01

    Visual phototransduction relies on the function of cyclic nucleotide-gated channels in the rod and cone photoreceptor outer segment plasma membranes. The role of these ion channels is to translate light-triggered changes in the second messenger cyclic guanosine 3'-5'-monophosphate levels into an electrical signal that is further processed within the retinal network and then sent to higher visual centers. Rod and cone photoreceptors express distinct CNG channels. The rod photoreceptor CNG channel is composed of one CNGB1 and three CNGA1 subunits, whereas the cone channel is formed by one CNGB3 and three CNGA3 subunits. Mutations in any of these channel subunits result in severe and currently untreatable retinal degenerative diseases like retinitis pigmentosa or achromatopsia. In this review, we provide an overview of the human diseases and relevant animal models of CNG channelopathies. Furthermore, we summarize recent results from preclinical gene therapy studies using adeno-associated viral vectors and discuss the efficacy and translational potential of these gene therapeutic approaches.

  6. An Effective Counterselection System for Listeria monocytogenes and Its Use To Characterize the Monocin Genomic Region of Strain 10403S

    PubMed Central

    Argov, Tal; Rabinovich, Lev; Sigal, Nadejda

    2016-01-01

    ABSTRACT Construction of Listeria monocytogenes mutants by allelic exchange has been laborious and time-consuming due to lack of proficient selection markers for the final recombination event, that is, a marker conveying substance sensitivity to the bacteria bearing it, enabling the exclusion of merodiploids and selection for plasmid loss. In order to address this issue, we engineered a counterselection marker based on a mutated phenylalanyl-tRNA synthetase gene (pheS*). This mutation renders the phenylalanine-binding site of the enzyme more promiscuous and allows the binding of the toxic p-chloro-phenylalanine analog (p-Cl-phe) as a substrate. When pheS* is introduced into L. monocytogenes and highly expressed under control of a constitutively active promoter, the bacteria become sensitive to p-Cl-phe supplemented in the medium. This enabled us to utilize pheS* as a negative selection marker and generate a novel, efficient suicide vector for allelic exchange in L. monocytogenes. We used this vector to investigate the monocin genomic region in L. monocytogenes strain 10403S by constructing deletion mutants of the region. We have found this region to be active and to cause bacterial lysis upon mitomycin C treatment. The future applications of such an effective counterselection system, which does not require any background genomic alterations, are vast, as it can be modularly used in various selection systems (e.g., genetic screens). We expect this counterselection marker to be a valuable genetic tool in research on L. monocytogenes. IMPORTANCE L. monocytogenes is an opportunistic intracellular pathogen and a widely studied model organism. An efficient counterselection marker is a long-standing need in Listeria research for improving the ability to design and perform various genetic manipulations and screening systems for different purposes. We report the construction and utilization of an efficient suicide vector for allelic exchange which can be conjugated, leaves

  7. An Effective Counterselection System for Listeria monocytogenes and Its Use To Characterize the Monocin Genomic Region of Strain 10403S.

    PubMed

    Argov, Tal; Rabinovich, Lev; Sigal, Nadejda; Herskovits, Anat A

    2017-03-15

    Construction of Listeria monocytogenes mutants by allelic exchange has been laborious and time-consuming due to lack of proficient selection markers for the final recombination event, that is, a marker conveying substance sensitivity to the bacteria bearing it, enabling the exclusion of merodiploids and selection for plasmid loss. In order to address this issue, we engineered a counterselection marker based on a mutated phenylalanyl-tRNA synthetase gene (pheS*). This mutation renders the phenylalanine-binding site of the enzyme more promiscuous and allows the binding of the toxic p-chloro-phenylalanine analog (p-Cl-phe) as a substrate. When pheS* is introduced into L. monocytogenes and highly expressed under control of a constitutively active promoter, the bacteria become sensitive to p-Cl-phe supplemented in the medium. This enabled us to utilize pheS* as a negative selection marker and generate a novel, efficient suicide vector for allelic exchange in L. monocytogenes We used this vector to investigate the monocin genomic region in L. monocytogenes strain 10403S by constructing deletion mutants of the region. We have found this region to be active and to cause bacterial lysis upon mitomycin C treatment. The future applications of such an effective counterselection system, which does not require any background genomic alterations, are vast, as it can be modularly used in various selection systems (e.g., genetic screens). We expect this counterselection marker to be a valuable genetic tool in research on L. monocytogenesIMPORTANCEL. monocytogenes is an opportunistic intracellular pathogen and a widely studied model organism. An efficient counterselection marker is a long-standing need in Listeria research for improving the ability to design and perform various genetic manipulations and screening systems for different purposes. We report the construction and utilization of an efficient suicide vector for allelic exchange which can be conjugated, leaves no marker in

  8. [Gene replacement therapy in achromatopsia type 2].

    PubMed

    Mühlfriedel, R; Tanimoto, N; Seeliger, M W

    2014-03-01

    Achromatopsia is an autosomal recessive inherited retinal disease caused by a complete loss of cone photoreceptor function. About 80 % of achromatopsia patients show mutations in the alpha or beta subunit (A3 and B3) of the cGMP controlled cation channel CNG (cyclic nucleotide-gated channel) of cone photoreceptors. Homologous to the human disease, CNGA3 deficient mice reveal a loss of cone specific functionality leading to degeneration of affected cone photoreceptors. The Institute for Ophthalmic Research in Tübingen has now succeeded in curing achromatopsia ACHM2 in an animal model. In this article, we explain the recombinant adeno-associated virus-based approach in detail. Furthermore, applied non-invasive diagnostic techniques for quality and success control, ERG, SLO and OCT, are described. The success of the therapy is indicated by a restored cone photoreceptor function as well as the neuronal processing of retinal signals resulting in a specific, cone-mediated behaviour. The outstanding results derived from the animal model are the starting point for the first human translation of a gene therapy for achromatopsia in Germany.

  9. Nonhomologous end joining-mediated gene replacement in plant cells.

    PubMed

    Weinthal, Dan Michael; Taylor, Roslyn Ann; Tzfira, Tzvi

    2013-05-01

    Stimulation of the homologous recombination DNA-repair pathway via the induction of genomic double-strand breaks (DSBs) by zinc finger nucleases (ZFNs) has been deployed for gene replacement in plant cells. Nonhomologous end joining (NHEJ)-mediated repair of DSBs, on the other hand, has been utilized for the induction of site-specific mutagenesis in plants. Since NHEJ is the dominant DSB repair pathway and can also lead to the capture of foreign DNA molecules, we suggest that it can also be deployed for gene replacement. An acceptor DNA molecule in which a green fluorescent protein (GFP) coding sequence (gfp) was flanked by ZFN recognition sequences was used to produce transgenic target plants. A donor DNA molecule in which a promoterless hygromycin B phosphotransferase-encoding gene (hpt) was flanked by ZFN recognition sequences was constructed. The donor DNA molecule and ZFN expression cassette were delivered into target plants. ZFN-mediated site-specific mutagenesis and complete removal of the GFP coding sequence resulted in the recovery of hygromycin-resistant plants that no longer expressed GFP and in which the hpt gene was unlinked to the acceptor DNA. More importantly, ZFN-mediated digestion of both donor and acceptor DNA molecules resulted in NHEJ-mediated replacement of the gfp with hpt and recovery of hygromycin-resistant plants that no longer expressed GFP and in which the hpt gene was physically linked to the acceptor DNA. Sequence and phenotypical analyses, and transmission of the replacement events to the next generation, confirmed the stability of the NHEJ-induced gene exchange, suggesting its use as a novel method for transgene replacement and gene stacking in plants.

  10. Targeted Curing of All Lysogenic Bacteriophage from Streptococcus pyogenes Using a Novel Counter-selection Technique.

    PubMed

    Euler, Chad W; Juncosa, Barbara; Ryan, Patricia A; Deutsch, Douglas R; McShan, W Michael; Fischetti, Vincent A

    2016-01-01

    Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (KanR) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (SmS), into a targeted prophage on the chromosome of a streptomycin resistant (SmR) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the KanS/SmR phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1ΔΦ, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1ΔΦ to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and

  11. Developing Universal Genetic Tools for Rapid and Efficient Deletion Mutation in Vibrio Species Based on Suicide T-Vectors Carrying a Novel Counterselectable Marker, vmi480

    PubMed Central

    Luo, Peng; He, Xiangyan; Liu, Qiuting; Hu, Chaoqun

    2015-01-01

    Despite that Vibrio spp. have a significant impact on the health of humans and aquatic animals, the molecular basis of their pathogenesis is little known, mainly due to the limited genetic tools for the functional research of genes in Vibrio. In some cases, deletion of target DNAs in Vibrio can be achieved through the use of suicide vectors. However, these strategies are time-consuming and lack universality, and the widely used counterselectable gene sacB does not work well in Vibrio cells. In this study, we developed universal genetic tools for rapid and efficient deletion mutations in Vibrio species based on suicide T-Vectors carrying a novel counterselectable marker, vmi480. We explored two uncharacterized genes, vmi480 and vmi470, in a genomic island from Vibrio mimicus VM573 and confirmed that vmi480 and vmi470 constitute a two-component toxin-antitoxin system through deletion and expression of vmi480 and vmi470. The product of vmi480 exhibited strong toxicity to Escherichia coli cells. Based on vmi480 and the PBAD or PTAC promoter system, we constructed two suicide T-vectors, pLP11 and pLP12, and each of these vectors contained a multiple cloning region with two AhdI sites. Both vectors linearized by AhdI digestion could be stored and directly ligated with purified PCR products without a digestion step. By using pLP11 and pLP12 coupled with a highly efficient conjugation system provided by E. coli β2163, six genes from four representative Vibrio species were easily deleted. By using the counterselective marker vmi480, we obtained 3–12 positive colonies (deletion mutants) among no more than 20 colonies randomly selected on counterselection plates. The strategy does not require the digestion of PCR products and suicide vectors every time, and it avoids large-scale screening colonies on counterselective plates. These results demonstrate that we successfully developed universal genetic tools for rapid and efficient gene deletion in Vibrio species. PMID

  12. Developing Universal Genetic Tools for Rapid and Efficient Deletion Mutation in Vibrio Species Based on Suicide T-Vectors Carrying a Novel Counterselectable Marker, vmi480.

    PubMed

    Luo, Peng; He, Xiangyan; Liu, Qiuting; Hu, Chaoqun

    2015-01-01

    Despite that Vibrio spp. have a significant impact on the health of humans and aquatic animals, the molecular basis of their pathogenesis is little known, mainly due to the limited genetic tools for the functional research of genes in Vibrio. In some cases, deletion of target DNAs in Vibrio can be achieved through the use of suicide vectors. However, these strategies are time-consuming and lack universality, and the widely used counterselectable gene sacB does not work well in Vibrio cells. In this study, we developed universal genetic tools for rapid and efficient deletion mutations in Vibrio species based on suicide T-Vectors carrying a novel counterselectable marker, vmi480. We explored two uncharacterized genes, vmi480 and vmi470, in a genomic island from Vibrio mimicus VM573 and confirmed that vmi480 and vmi470 constitute a two-component toxin-antitoxin system through deletion and expression of vmi480 and vmi470. The product of vmi480 exhibited strong toxicity to Escherichia coli cells. Based on vmi480 and the PBAD or PTAC promoter system, we constructed two suicide T-vectors, pLP11 and pLP12, and each of these vectors contained a multiple cloning region with two AhdI sites. Both vectors linearized by AhdI digestion could be stored and directly ligated with purified PCR products without a digestion step. By using pLP11 and pLP12 coupled with a highly efficient conjugation system provided by E. coli β2163, six genes from four representative Vibrio species were easily deleted. By using the counterselective marker vmi480, we obtained 3-12 positive colonies (deletion mutants) among no more than 20 colonies randomly selected on counterselection plates. The strategy does not require the digestion of PCR products and suicide vectors every time, and it avoids large-scale screening colonies on counterselective plates. These results demonstrate that we successfully developed universal genetic tools for rapid and efficient gene deletion in Vibrio species.

  13. Versatile dual-technology system for markerless allele replacement in Burkholderia pseudomallei.

    PubMed

    López, Carolina M; Rholl, Drew A; Trunck, Lily A; Schweizer, Herbert P

    2009-10-01

    Burkholderia pseudomallei is the etiologic agent of melioidosis, a rare but serious tropical disease. In the United States, genetic research with this select agent bacterium is strictly regulated. Although several select agent compliant methods have been developed for allelic replacement, all of them suffer from some drawbacks, such as a need for specific host backgrounds or use of minimal media. Here we describe a versatile select agent compliant allele replacement system for B. pseudomallei based on a mobilizable vector, pEXKm5, which contains (i) a multiple cloning site within a lacZalpha gene for facile cloning of recombinant DNA fragments, (ii) a constitutively expressed gusA indicator gene for visual detection of merodiploid formation and resolution, and (iii) elements required for resolution of merodiploids using either I-SceI homing endonuclease-stimulated recombination or sacB-based counterselection. The homing endonuclease-based allele replacement system is completed by pBADSce, which contains an araC-P(BAD)-I-sceI expression cassette for arabinose-inducible I-SceI expression and a temperature-sensitive pRO1600 replicon for facile plasmid curing. Complementing these systems is the improved Deltaasd Escherichia coli mobilizer strain RHO3. This strain is susceptible to commonly used antibiotics and allows nutritional counterselection on rich media because of its diaminopimelic acid auxotrophy. The versatility of the I-SceI- and sacB-based methods afforded by pEXKm5 in conjunction with E. coli RHO3 was demonstrated by isolation of diverse deletion mutants in several clinical, environmental, and laboratory B. pseudomallei strains. Finally, sacB-based counterselection was employed to isolate a defined chromosomal fabD(Ts) allele that causes synthesis of a temperature-sensitive FabD, an essential fatty acid biosynthesis enzyme.

  14. Counter-selectable marker for bacterial-based interaction trap systems.

    PubMed

    Meng, Xiangdong; Smith, Robin M; Giesecke, Astrid V; Joung, J Keith; Wolfe, Scot A

    2006-02-01

    Counter-selectable markers can be used in two-hybrid systems to search libraries for a protein or compound that interferes with a macromolecular interaction or to identify macromolecules from a population that cannot mediate a particular interaction. In this report, we describe the adaptation of the yeast URA3/5-FOA counter-selection system for use in bacterial interaction trap experiments. Two different URA3 reporter systems were developed that allow robust counter-selection: (i) a single copy F' episome reporter and (ii) a co-cistronic HIS3-URA3 reporter vector. The HIS3-URA3 reporter can be used for either positive or negative selections in appropriate bacterial strains. These reagents extend the utility of the bacterial two-hybrid system as an alternative to its yeast-based counterpart.

  15. Gene replacement and expression of foreign DNA in mycobacteria.

    PubMed Central

    Husson, R N; James, B E; Young, R A

    1990-01-01

    A system that permits molecular genetic manipulation of mycobacteria was developed on the basis of the yeast paradigm of gene replacement by homologous recombination. A shuttle vector that can replicate autonomously at a high copy number in Escherichia coli but must integrate into homologous DNA for survival in Mycobacterium smegmatis was constructed. The vector contains a ColE1 origin of replication, antibiotic resistance markers for ampicillin and kanamycin, a nutritional marker (pyrF) that allows both positive and negative selection in E. coli and M. smegmatis, and unique restriction sites that permit insertion of foreign DNA. Transformation of mycobacteria with this vector results in integration of its DNA into the genomic pyrF locus by either a single or a double homologous recombination event. With this system, the 65-kilodalton Mycobacterium leprae stress protein antigen was inserted into the M. smegmatis genome and expressed. This gene replacement technology, together with a uniquely useful pyrF marker, should be valuable for investigating mycobacterial pathobiology, for the development of candidate mycobacterial vaccine vehicles, and as a model for the development of molecular genetic systems in other pathogenic microorganisms. Images FIG. 2 FIG. 3 PMID:2153655

  16. Effects of G-gene Deletion and Replacement on Rabies Virus Vector Gene Expression

    PubMed Central

    Sato, Sho; Ohara, Shinya; Tsutsui, Ken-Ichiro; Iijima, Toshio

    2015-01-01

    The glycoprotein-gene (G gene) -deleted rabies virus (RV) vector is a powerful tool to examine the function and structure of neural circuits. We previously reported that the deletion of the G gene enhances the transgene expression level of the RV vector. However, the mechanism of this enhancement remains to be clarified. We presume that there are two possible factors for this enhancement. The first factor is the glycoprotein of RV, which shows cytotoxicity; thus, may cause a dysfunction in the translation process of infected cells. The second possible factor is the enhanced expression of the L gene, which encodes viral RNA polymerase. In the RV, it is known that the gene expression level is altered depending on the position of the gene. Since G-gene deletion displaces the L gene in the genome, the expression of the L gene and viral transcription may be enhanced. In this study, we compared the transgene expression level and viral transcription of three recombinant RV vectors. The effect of glycoprotein was examined by comparing the viral gene expression of G-gene-intact RV and G-gene-replaced RV. Despite the fact that the L-gene transcription level of these two RV vectors was similar, the G-gene-replaced RV vector showed higher viral transcription and transgene expression level than the G-gene-intact RV vector. To examine the effect of the position of the L gene, we compared the viral gene expression of the G-gene-deleted RV and G-gene-replaced RV. The G-gene-deleted RV vector showed higher L-gene transcription, viral transcription, and transgene expression level than the G-gene-replaced RV vector. These results indicate that G-gene deletion enhances the transgene expression level through at least two factors, the absence of glycoprotein and enhancement of L-gene expression. These findings enable investigators to design a useful viral vector that shows a controlled desirable transgene expression level in applications. PMID:26023771

  17. Light chain replacement: a new model for antibody gene rearrangement

    PubMed Central

    1995-01-01

    A functional B cell antigen receptor is thought to regulate antibody gene rearrangement either by stopping further rearrangement (exclusion) or by promoting additional rearrangement (editing). We have developed a new model to study the regulation of antibody gene rearrangement. In this model, we used gene targeting to replace the J kappa region with a functional V kappa-J kappa light chain gene. Two different strains of mice were created; one, V kappa 4R, has a V kappa 4-J kappa 4 rearrangement followed by a downstream J kappa 5 segment, while the other, V kappa 8R, has a V kappa 8-J kappa 5 light chain. Here, we analyze the influence of these functional light chains on light chain rearrangement. We show that some V kappa 4R and V kappa 8R B cells only have the V kappa R light chain rearrangement, whereas others undergo additional rearrangements. Additional rearrangement can occur not only at the other kappa allele or isotype (lambda), but also at the targeted locus in both V kappa 4R and V kappa 8R. Rearrangement to the downstream J kappa 5 segment is observed in V kappa 4R, as is deletion of the targeted locus in both V kappa 4R and V kappa 8R. The V kappa R models illustrate that a productively rearranged light chain can either terminate further rearrangement or allow further rearrangement. We attribute the latter to editing of autoantibodies and to corrections of dysfunctional receptors. PMID:7629511

  18. Gene replacement in Halobacterium halobium and expression of bacteriorhodopsin mutants.

    PubMed

    Krebs, M P; Mollaaghababa, R; Khorana, H G

    1993-03-01

    A gene replacement method has been developed to express bacteriorhodopsin mutants in the archaeon Halobacterium halobium. Selectable plasmids carrying the bacterioopsin gene (bop) were integrated at the chromosomal bop locus of H. halobium. Under nonselective conditions, recombinants were isolated that had lost the integrated plasmid and retained a single chromosomal copy of the bop gene. This approach was used to construct a bop deletion strain. By using this strain, recombinants were obtained that express wild-type bacteriorhodopsin and mutants known to be defective in proton translocation. The expressed proteins were purified in a membrane fraction similar to purple membrane and were characterized in this form. UV/visible spectra of dark- and light-adapted bacteriorhodopsin from wild-type and Asp-96 mutants were identical to those of purple membrane. Arg-82, Asp-85, and Asp-212 mutants had 10- to 50-nm red shifts in their absorption maxima and showed altered light adaptation. The proton translocation activity of the wild-type samples and purple membrane was comparable, whereas the mutants had 0-60% of wild-type activity. These results support earlier studies of proton translocation mutants expressed in Escherichia coli.

  19. Gene replacement in Halobacterium halobium and expression of bacteriorhodopsin mutants.

    PubMed Central

    Krebs, M P; Mollaaghababa, R; Khorana, H G

    1993-01-01

    A gene replacement method has been developed to express bacteriorhodopsin mutants in the archaeon Halobacterium halobium. Selectable plasmids carrying the bacterioopsin gene (bop) were integrated at the chromosomal bop locus of H. halobium. Under nonselective conditions, recombinants were isolated that had lost the integrated plasmid and retained a single chromosomal copy of the bop gene. This approach was used to construct a bop deletion strain. By using this strain, recombinants were obtained that express wild-type bacteriorhodopsin and mutants known to be defective in proton translocation. The expressed proteins were purified in a membrane fraction similar to purple membrane and were characterized in this form. UV/visible spectra of dark- and light-adapted bacteriorhodopsin from wild-type and Asp-96 mutants were identical to those of purple membrane. Arg-82, Asp-85, and Asp-212 mutants had 10- to 50-nm red shifts in their absorption maxima and showed altered light adaptation. The proton translocation activity of the wild-type samples and purple membrane was comparable, whereas the mutants had 0-60% of wild-type activity. These results support earlier studies of proton translocation mutants expressed in Escherichia coli. Images Fig. 2 Fig. 3 Fig. 6 PMID:8446619

  20. Marker recycling via 5-fluoroorotic acid and 5-fluorocytosine counter-selection in the white-rot agaricomycete Pleurotus ostreatus.

    PubMed

    Nakazawa, Takehito; Tsuzuki, Masami; Irie, Toshikazu; Sakamoto, Masahiro; Honda, Yoichi

    2016-09-01

    Of all of the natural polymers, lignin, an aromatic heteropolymer in plant secondary cell walls, is the most resistant to biological degradation. White-rot fungi are the only known organisms that can depolymerize or modify wood lignin. Investigating the mechanisms underlying lignin biodegradation by white-rot fungi would contribute to the ecofriendly utilization of woody biomass as renewable resources in the future. Efficient gene disruption, which is generally very challenging in the white-rot fungi, was established in Pleurotus ostreatus (the oyster mushroom). Some of the genes encoding manganese peroxidases, enzymes that are considered to be involved in lignin biodegradation, were disrupted separately, and the phenotype of each single-gene disruptant was analysed. However, it remains difficult to generate multi-gene disruptants in this fungus. Here we developed a new genetic transformation marker in P. ostreatus and demonstrated two marker recycling methods that use counter-selection to generate a multigene disruptant. This study will enable future genetic studies of white-rot fungi, and it will increase our understanding of the complicated mechanisms, which involve various enzymes, including lignin-degrading enzymes, underlying lignin biodegradation by these fungi. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  1. Auxotrophic markers pyrF and proC can replace antibiotic markers on protein production plasmids in high-cell-density Pseudomonas fluorescens fermentation.

    PubMed

    Schneider, Jane C; Jenings, Annika F; Mun, Deborah M; McGovern, Patricia M; Chew, Lawrence C

    2005-01-01

    The use of antibiotic-resistance genes as selectable markers in transgenic organisms is coming under increased scrutiny, for fear that they may spread to human pathogens, thereby reducing the effectiveness of antibiotic therapy. A current Pseudomonas fluorescens protein expression system uses a tetracycline resistance gene (tetR/tetA) to maintain an expression plasmid under control of a repressible promoter and a kanamycin resistance gene (kanR) to maintain a plasmid carrying a repressor gene. We investigated using auxotrophic markers to replace these two antibiotic resistance genes: pyrF (encoding orotidine-5'-phosphate decarboxylase) in place of tetR/tetA and proC (encoding pyrroline-5-carboxylate reductase) in place of kanR, complementing their respective precise chromosomal deletions created by allele exchange using a suicide vector carrying pyrF as a counterselectable marker. The resulting strains, devoid of antibiotic-resistance genes, were shown to achieve high productivity of nitrilase and thermostable alpha-amylase equal to that of the former antibiotic-resistant production host. The production plasmids were stable. The pyrF (uracil-dependent) background of the production host strain also allows us to sequentially alter the genome to incorporate other desired genomic changes, deletions, or insertions using 5'-fluoroorotic acid counterselection, restoring the selectable marker after each step.

  2. High-efficiency genome editing and allele replacement in prototrophic and wild strains of Saccharomyces.

    PubMed

    Alexander, William G; Doering, Drew T; Hittinger, Chris Todd

    2014-11-01

    Current genome editing techniques available for Saccharomyces yeast species rely on auxotrophic markers, limiting their use in wild and industrial strains and species. Taking advantage of the ancient loss of thymidine kinase in the fungal kingdom, we have developed the herpes simplex virus thymidine kinase gene as a selectable and counterselectable marker that forms the core of novel genome engineering tools called the H: aploid E: ngineering and R: eplacement P: rotocol (HERP) cassettes. Here we show that these cassettes allow a researcher to rapidly generate heterogeneous populations of cells with thousands of independent chromosomal allele replacements using mixed PCR products. We further show that the high efficiency of this approach enables the simultaneous replacement of both alleles in diploid cells. Using these new techniques, many of the most powerful yeast genetic manipulation strategies are now available in wild, industrial, and other prototrophic strains from across the diverse Saccharomyces genus. Copyright © 2014 by the Genetics Society of America.

  3. Dystrophin Gene Replacement and Gene Repair Therapy for Duchenne Muscular Dystrophy in 2016: An Interview

    PubMed Central

    Duan, Dongsheng

    2016-01-01

    After years of relentless efforts, gene therapy has now begun to deliver its therapeutic promise in several diseases. A number of gene therapy products have received regulatory approval in Europe and Asia. Duchenne muscular dystrophy (DMD) is an X-linked inherited lethal muscle disease. It is caused by mutations in the dystrophin gene. Replacing and/or repairing the mutated dystrophin gene holds great promises to treated DMD at the genetic level. Last several years have evidenced significant developments in preclinical experimentations in murine and canine models of DMD. There has been a strong interest in moving these promising findings to clinical trials. In light of rapid progress in this field, the Parent Project Muscular Dystrophy (PPMD) recently interviewed me on the current status of DMD gene therapy and readiness for clinical trials. Here I summarized the interview with PPMD. PMID:27003751

  4. Dystrophin gene replacement and gene repair therapy for Duchenne muscular dystrophy in 2016.

    PubMed

    Duan, Dongsheng

    2016-03-04

    After years of relentless efforts, gene therapy has now begun to deliver its therapeutic promise in several diseases. A number of gene therapy products have received regulatory approval in Europe and Asia. Duchenne muscular dystrophy (DMD) is an X-linked inherited lethal muscle disease. It is caused by mutations in the dystrophin gene. Replacing and/or repair the mutated dystrophin gene holds great promises to treated DMD at the genetic level. Last several years have evidenced significant developments in preclinical experimentations in murine and canine models of DMD. There has been a strong interest in moving these promising findings to clinical trials. In light of rapid progress in this field, the Parent Project Muscular Dystrophy (PPMD) recently interviewed me on the current status of DMD gene therapy. Here I summarized the interview with PPMD.

  5. Dystrophin Gene Replacement and Gene Repair Therapy for Duchenne Muscular Dystrophy in 2016: An Interview.

    PubMed

    Duan, Dongsheng

    2016-03-01

    After years of relentless efforts, gene therapy has now begun to deliver its therapeutic promise in several diseases. A number of gene therapy products have received regulatory approval in Europe and Asia. Duchenne muscular dystrophy (DMD) is an X-linked inherited lethal muscle disease. It is caused by mutations in the dystrophin gene. Replacing and/or repairing the mutated dystrophin gene holds great promises to treated DMD at the genetic level. Last several years have evidenced significant developments in preclinical experimentations in murine and canine models of DMD. There has been a strong interest in moving these promising findings to clinical trials. In light of rapid progress in this field, the Parent Project Muscular Dystrophy (PPMD) recently interviewed me on the current status of DMD gene therapy and readiness for clinical trials. Here I summarized the interview with PPMD.

  6. Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus.

    PubMed Central

    Mollet, B; Knol, J; Poolman, B; Marciset, O; Delley, M

    1993-01-01

    Several pGEM5- and pUC19-derived plasmids containing a selectable erythromycin resistance marker were integrated into the chromosome of Streptococcus thermophilus at the loci of the lactose-metabolizing genes. Integration occurred via homologous recombination and resulted in cointegrates between plasmid and genome, flanked by the homologous DNA used for integration. Selective pressure on the plasmid-located erythromycin resistance gene resulted in multiple amplifications of the integrated plasmid. Release of this selective pressure, however, gave way to homologous resolution of the cointegrate structures. By integration and subsequent resolution, we were able to replace the chromosomal lacZ gene with a modified copy carrying an in vitro-generated deletion. In the same way, we integrated a promoterless chloramphenicol acetyltransferase (cat) gene between the chromosomal lacS and lacZ genes of the lactose operon. The inserted cat gene became a functional part of the operon and was expressed and regulated accordingly. Selective pressure on the essential lacS and lacZ genes under normal growth conditions in milk ensures the maintenance and expression of the integrated gene. As there are only minimal repeated DNA sequences (an NdeI site) flanking the inserted cat gene, it was stably maintained even in the absence of lactose, i.e., when grown on sucrose or glucose. The methodology represents a stable system in which to express and regulate foreign genes in S. thermophilus, which could qualify in the future for an application with food. Images PMID:8331064

  7. VH Replacement Footprint Analyzer-I, a Java-Based Computer Program for Analyses of Immunoglobulin Heavy Chain Genes and Potential VH Replacement Products in Human and Mouse.

    PubMed

    Huang, Lin; Lange, Miles D; Zhang, Zhixin

    2014-01-01

    VH replacement occurs through RAG-mediated secondary recombination between a rearranged VH gene and an upstream unrearranged VH gene. Due to the location of the cryptic recombination signal sequence (cRSS, TACTGTG) at the 3' end of VH gene coding region, a short stretch of nucleotides from the previous rearranged VH gene can be retained in the newly formed VH-DH junction as a "footprint" of VH replacement. Such footprints can be used as markers to identify Ig heavy chain (IgH) genes potentially generated through VH replacement. To explore the contribution of VH replacement products to the antibody repertoire, we developed a Java-based computer program, VH replacement footprint analyzer-I (VHRFA-I), to analyze published or newly obtained IgH genes from human or mouse. The VHRFA-1 program has multiple functional modules: it first uses service provided by the IMGT/V-QUEST program to assign potential VH, DH, and JH germline genes; then, it searches for VH replacement footprint motifs within the VH-DH junction (N1) regions of IgH gene sequences to identify potential VH replacement products; it can also analyze the frequencies of VH replacement products in correlation with publications, keywords, or VH, DH, and JH gene usages, and mutation status; it can further analyze the amino acid usages encoded by the identified VH replacement footprints. In summary, this program provides a useful computation tool for exploring the biological significance of VH replacement products in human and mouse.

  8. Immunoglobulin V gene replacement is caused by the intramolecular DNA deletion mechanism.

    PubMed Central

    Usuda, S; Takemori, T; Matsuoka, M; Shirasawa, T; Yoshida, K; Mori, A; Ishizaka, K; Sakano, H

    1992-01-01

    Circular DNA resulting from V gene replacement was studied with an A-MuLV transformed cell line containing ablts. This cell line undergoes V gene replacement at elevated temperatures in the immunoglobulin (Ig) heavy chain (H) gene. Examination of circular DNA revealed that a heptamer-related sequence (TACTGTG) within the coding region of VDJ was joined to the recombination signal sequence (RSS) of a germline VH segment. This provides direct evidence for a intramolecular DNA deletion mechanism for V gene replacement. In the pre-B cell line as well as in in vivo lymphocytes, unusual circular DNAs were found which were structurally similar to the V gene replacement circles. They represented excision products of the deletion type recombination between one complete RSS and a heptamer-like sequence in the Ig H region. PMID:1311252

  9. Mitochondrial gene replacement in primate offspring and embryonic stem cells.

    PubMed

    Tachibana, Masahito; Sparman, Michelle; Sritanaudomchai, Hathaitip; Ma, Hong; Clepper, Lisa; Woodward, Joy; Li, Ying; Ramsey, Cathy; Kolotushkina, Olena; Mitalipov, Shoukhrat

    2009-09-17

    Mitochondria are found in all eukaryotic cells and contain their own genome (mitochondrial DNA or mtDNA). Unlike the nuclear genome, which is derived from both the egg and sperm at fertilization, the mtDNA in the embryo is derived almost exclusively from the egg; that is, it is of maternal origin. Mutations in mtDNA contribute to a diverse range of currently incurable human diseases and disorders. To establish preclinical models for new therapeutic approaches, we demonstrate here that the mitochondrial genome can be efficiently replaced in mature non-human primate oocytes (Macaca mulatta) by spindle-chromosomal complex transfer from one egg to an enucleated, mitochondrial-replete egg. The reconstructed oocytes with the mitochondrial replacement were capable of supporting normal fertilization, embryo development and produced healthy offspring. Genetic analysis confirmed that nuclear DNA in the three infants born so far originated from the spindle donors whereas mtDNA came from the cytoplast donors. No contribution of spindle donor mtDNA was detected in offspring. Spindle replacement is shown here as an efficient protocol replacing the full complement of mitochondria in newly generated embryonic stem cell lines. This approach may offer a reproductive option to prevent mtDNA disease transmission in affected families.

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

    PubMed

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

    2011-08-01

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

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

    SciTech Connect

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

    2011-08-08

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

  12. A diaminopimelic acid auxotrophic Escherichia coli donor provides improved counterselection following intergeneric conjugation with actinomycetes.

    PubMed

    Allard, Nancy; Garneau, Daniel; Poulin-Laprade, Dominic; Burrus, Vincent; Brzezinski, Ryszard; Roy, Sébastien

    2015-08-01

    Considering the medical, biotechnological, and economical importance of actinobacteria, there is a continuous need to improve the tools for genetic engineering of a broad range of these microorganisms. Intergeneric conjugation has proven to be a valuable yet imperfect tool for this purpose. The natural resistance of many actinomycetes to nalidixic acid (Nal) is generally exploited to eliminate the sensitive Escherichia coli donor strain following conjugation. Nevertheless, Nal can delay growth and have other unexpected effects on the recipient strain. To provide an improved alternative to antibiotics, we propose a postconjugational counterselection using a diaminopimelic acid (DAP) auxotrophic donor strain. The DAP-negative phenotype was obtained by introducing a dapA deletion into the popular methylase-negative donor strain E. coli ET12567/pUZ8002. The viability of ET12567 and its ΔdapA mutant exposed to DAP deprivation or Nal selection were compared in liquid pure culture and after mating with Streptomyces coelicolor. Results showed that death of the E. coli ΔdapA Nal-sensitive donor strain occurred more efficiently when subjected to DAP deprivation than when exposed to Nal. Our study shows that postconjugational counterselection based on DAP deprivation circumvents the use of antibiotics and will facilitate the transfer of plasmids into actinomycetes with high biotechnological potential, yet currently not accessible to conjugative techniques.

  13. VH Replacement Footprint Analyzer-I, a Java-Based Computer Program for Analyses of Immunoglobulin Heavy Chain Genes and Potential VH Replacement Products in Human and Mouse

    PubMed Central

    Huang, Lin; Lange, Miles D.; Zhang, Zhixin

    2014-01-01

    VH replacement occurs through RAG-mediated secondary recombination between a rearranged VH gene and an upstream unrearranged VH gene. Due to the location of the cryptic recombination signal sequence (cRSS, TACTGTG) at the 3′ end of VH gene coding region, a short stretch of nucleotides from the previous rearranged VH gene can be retained in the newly formed VH–DH junction as a “footprint” of VH replacement. Such footprints can be used as markers to identify Ig heavy chain (IgH) genes potentially generated through VH replacement. To explore the contribution of VH replacement products to the antibody repertoire, we developed a Java-based computer program, VH replacement footprint analyzer-I (VHRFA-I), to analyze published or newly obtained IgH genes from human or mouse. The VHRFA-1 program has multiple functional modules: it first uses service provided by the IMGT/V-QUEST program to assign potential VH, DH, and JH germline genes; then, it searches for VH replacement footprint motifs within the VH–DH junction (N1) regions of IgH gene sequences to identify potential VH replacement products; it can also analyze the frequencies of VH replacement products in correlation with publications, keywords, or VH, DH, and JH gene usages, and mutation status; it can further analyze the amino acid usages encoded by the identified VH replacement footprints. In summary, this program provides a useful computation tool for exploring the biological significance of VH replacement products in human and mouse. PMID:24575092

  14. Reciprocal gene replacements reveal unique functions for Phox2 genes during neural differentiation

    PubMed Central

    Coppola, Eva; Pattyn, Alexandre; Guthrie, Sarah C; Goridis, Christo; Studer, Michèle

    2005-01-01

    The paralogous paired-like homeobox genes Phox2a and Phox2b are involved in the development of specific neural subtypes in the central and peripheral nervous systems. The different phenotypes of Phox2 knockout mutants, together with their asynchronous onset of expression, prompted us to generate two knock-in mutant mice, in which Phox2a is replaced by the Phox2b coding sequence, and vice versa. Our results indicate that Phox2a and Phox2b are not functionally equivalent, as only Phox2b can fulfill the role of Phox2a in the structures that depend on both genes. Furthermore, we demonstrate unique roles of Phox2 genes in the differentiation of specific motor neurons. Whereas the oculomotor and the trochlear neurons require Phox2a for their proper development, the migration of the facial branchiomotor neurons depends on Phox2b. Therefore, our analysis strongly indicates that biochemical differences between the proteins rather than temporal regulation of their expression account for the specific function of each paralogue. PMID:16319924

  15. Gene replacement therapy for sickle cell disease and other blood disorders.

    PubMed

    Townes, Tim M

    2008-01-01

    Previous studies have demonstrated that sickle cell disease (SCD) can be corrected in mouse models by transduction of hematopoietic stem cells with lentiviral vectors containing anti-sickling globin genes followed by transplantation of these cells into syngeneic recipients. Although self-inactivating (SIN) lentiviral vectors with or without insulator elements should provide a safe and effective treatment in humans, some concerns about insertional mutagenesis persist. An ideal correction would involve replacement of the sickle globin gene (betaS) with a normal copy of the gene (betaA). We recently derived embryonic stem (ES) cells from a novel knockin mouse model of SCD and tested a protocol for correcting the sickle mutation by homologous recombination. Animals derived after gene replacement produced high levels of normal human hemoglobin (HbA), and the pathology associated with SCD was corrected. These experiments provided a foundation for similar studies in which our group collaborated with Rudolf Jaenisch's laboratory to correct SCD by gene replacement in iPS (induced pluripotent stem) cells derived by direct reprogramming of sickle skin fibroblasts. Corrected iPS cells were differentiated into hematopoeitic progenitors that were transplanted into irradiated sickle recipients. The transplanted animals produced high levels of normal human HbA, and the pathology of SCD was corrected. These proof-of-principle studies provide a foundation for the development of gene replacement therapy for human patients with SCD and other blood disorders.

  16. Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement

    PubMed Central

    Clauwers, Charlien; Vanoirbeek, Kristof; Delbrassinne, Laurence

    2016-01-01

    ABSTRACT Group II nonproteolytic Clostridium botulinum (gIICb) strains are an important concern for the safety of minimally processed ready-to-eat foods, because they can grow and produce botulinum neurotoxin during refrigerated storage. The principles of control of gIICb by conventional food processing and preservation methods have been well investigated and translated into guidelines for the food industry; in contrast, the effectiveness of emerging processing and preservation techniques has been poorly documented. The reason is that experimental studies with C. botulinum are cumbersome because of biosafety and biosecurity concerns. In the present work, we report the construction of two nontoxigenic derivatives of the type E gIICb strain NCTC 11219. In the first strain, the botulinum toxin gene (bont/E) was insertionally inactivated with a retargeted intron using the ClosTron system. In the second strain, bont/E was exchanged for an erythromycin resistance gene using a new gene replacement strategy that makes use of pyrE as a bidirectional selection marker. Growth under optimal and stressed conditions, sporulation efficiency, and spore heat resistance of the mutants were unaltered, except for small differences in spore heat resistance at 70°C and in growth at 2.3% NaCl. The mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with gIICb. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in gIICb and other clostridia. IMPORTANCE The nontoxigenic mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with psychrotrophic Clostridium botulinum. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in clostridia. PMID:26994073

  17. Use of rpsL for dominance selection and gene replacement in Streptomyces roseosporus.

    PubMed Central

    Hosted, T J; Baltz, R H

    1997-01-01

    We developed a gene replacement system using the rpsL gene of Streptomyces roseosporus and demonstrated its utility by constructing a deletion in the S. roseosporus glnA gene. A 1.3-kb BamHI fragment that hybridized to the Mycobacterium smegmatis rpsL gene was subcloned from an S. roseosporus cosmid library and sequenced. Plasmid pRHB514 containing the rpsL gene conferred streptomycin sensitivity (Sm(S)) to the Sm(r) S. roseosporus TH149. The temperature-sensitive plasmid pRHB543 containing rpsL and the S. roseosporus glnA gene disrupted with a hygromycin resistance (Hm(r)) gene was introduced into S. roseosporus TH149, and recombinants containing single and double crossovers were obtained after a temperature increase. Southern hybridization analysis revealed that single crossovers occurred in the glnA or rpsL genes and that double crossovers resulted in replacement of the chromosomal glnA gene with the disrupted glnA. Glutamine synthetase activity was undetectable in the recombinant containing the disrupted glnA gene. PMID:8981996

  18. High frequency vector-mediated transformation and gene replacement in Tetrahymena.

    PubMed Central

    Gaertig, J; Gu, L; Hai, B; Gorovsky, M A

    1994-01-01

    Recently, we developed a mass DNA-mediated transformation technique for the ciliated protozoan Tetrahymena thermophila that introduces transforming DNA by electroporation into conjugating cells. Other studies demonstrated that a neomycin resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when microinjected into the macronucleus. We describe the use of conjugant electrotransformation (CET) for gene replacement and for the development of new independently replicating vectors and a gene cassette that can be used as a selectable marker in gene knockout experiments. Using CET, the neomycin resistance gene flanked by H4-I sequences transformed Tetrahymena, resulting in the replacement of the H4-I gene or integrative recombination of the H4-I/neo/H4-I gene (but not vector sequences) in the 5' or 3' flanking region of the H4-I locus. Gene replacement was obtained with non-digested plasmid DNA but releasing the insert increased the frequency of replacement events about 6-fold. The efficiency of transformation by the H4-I/neo/H4-I selectable marker was unchanged when a single copy of the Tetrahymena rDNA replication origin was included on the transforming plasmid. However, the efficiency of transformation using CET increased greatly when a tandem repeat of the replication origin fragment was used. This high frequency of transformation enabled mapping of the region required for H4-I promoter function to within 333 bp upstream of the initiator ATG. Similarly approximately 300 bp of sequence downstream of the translation terminator TGA of the beta-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene. This hybrid H4-I/neo/BTU2 gene did not transform Tetrahymena when subcloned on a plasmid lacking an origin of replication, but did transform at high frequency on a two origin plasmid. Thus, the H4-I/neo/BTU2 cassette is a selectable marker that can be used for gene

  19. Gene replacement therapies for duchenne muscular dystrophy using adeno-associated viral vectors.

    PubMed

    Seto, Jane T; Ramos, Julian N; Muir, Lindsey; Chamberlain, Jeffrey S; Odom, Guy L

    2012-06-01

    The muscular dystrophies collectively represent a major health challenge, as few significant treatment options currently exist for any of these disorders. Recent years have witnessed a proliferation of novel approaches to therapy, spanning increased testing of existing and new pharmaceuticals, DNA delivery (both anti-sense oligonucleotides and plasmid DNA), gene therapies and stem cell technologies. While none of these has reached the point of being used in clinical practice, all show promise for being able to impact different types of muscular dystrophies. Our group has focused on developing direct gene replacement strategies to treat recessively inherited forms of muscular dystrophy, particularly Duchenne and Becker muscular dystrophy (DMD/BMD). Both forms of dystrophy are caused by mutations in the dystrophin gene and all cases can in theory be treated by gene replacement using synthetic forms of the dystrophin gene. The major challenges for success of this approach are the development of a suitable gene delivery shuttle, generating a suitable gene expression cassette able to be carried by such a shuttle, and achieving safe and effective delivery without elicitation of a destructive immune response. This review summarizes the current state of the art in terms of using adeno-associated viral vectors to deliver synthetic dystrophin genes for the purpose of developing gene therapy for DMD.

  20. Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells

    PubMed Central

    Byrne, Susan M.; Ortiz, Luis; Mali, Prashant; Aach, John; Church, George M.

    2015-01-01

    Sequence-specific nucleases such as TALEN and the CRISPR/Cas9 system have so far been used to disrupt, correct or insert transgenes at precise locations in mammalian genomes. We demonstrate efficient ‘knock-in’ targeted replacement of multi-kilobase genes in human induced pluripotent stem cells (iPSC). Using a model system replacing endogenous human genes with their mouse counterpart, we performed a comprehensive study of targeting vector design parameters for homologous recombination. A 2.7 kilobase (kb) homozygous gene replacement was achieved in up to 11% of iPSC without selection. The optimal homology arm length was around 2 kb, with homology length being especially critical on the arm not adjacent to the cut site. Homologous sequence inside the cut sites was detrimental to targeting efficiency, consistent with a synthesis-dependent strand annealing (SDSA) mechanism. Using two nuclease sites, we observed a high degree of gene excisions and inversions, which sometimes occurred more frequently than indel mutations. While homozygous deletions of 86 kb were achieved with up to 8% frequency, deletion frequencies were not solely a function of nuclease activity and deletion size. Our results analyzing the optimal parameters for targeting vector design will inform future gene targeting efforts involving multi-kilobase gene segments, particularly in human iPSC. PMID:25414332

  1. Double replacement gene targeting for the production of a series of mouse strains with different prion protein gene alterations

    SciTech Connect

    Moore, R.C.; Redhead, N.J.; Selfridge, J.

    1995-09-01

    We have developed a double replacement gene targeting strategy which enables the production of a series of mouse strains bearing different subtle alterations to endogenous genes. This is a two-step process in which a region of the gene of interest is first replaced with a selectable marker to produce an inactivated allele, which is then re-targeted with a second vector to reconstruct the inactivated allele, concomitantly introducing an engineered mutation. Five independent embryonic stem cell lines have been produced bearing different targeted alterations to the prion protein gene, including one which raises the level of expression. We have constructed mice bearing the codon 101 proline to leucine substitution linked to the human familial prion disease, Gerstmann-Straussler-Scheinker syndrome. We anticipate that this procedure will have applications to the study of human inherited diseases and the development of therapies. 43 refs., 6 figs., 1 tab.

  2. The treatment of hemophilia A: from protein replacement to AAV-mediated gene therapy.

    PubMed

    Youjin, Shen; Jun, Yin

    2009-03-01

    Factor VIII (FVIII) is an essential component in blood coagulation, a deficiency of which causes the serious bleeding disorder hemophilia A. Recently, with the development of purification level and recombinant techniques, protein replacement treatment to hemophiliacs is relatively safe and can prolong their life expectancy. However, because of the possibility of unknown contaminants in plasma-derived FVIII and recombinant FVIII, and high cost for hemophiliacs to use these products, gene therapy for hemophilia A is an attractive alternative to protein replacement therapy. Thus far, the adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. It is likely that hemophilia will be the first genetic disease to be cured by somatic cell gene therapy.

  3. Unparalleled replacement of native mitochondrial genes by foreign homologs in a holoparasitic plant.

    PubMed

    Sanchez-Puerta, M Virginia; García, Laura E; Wohlfeiler, Josefina; Ceriotti, Luis F

    2017-04-01

    Horizontal gene transfer (HGT) among flowering plant mitochondria occurs frequently and, in most cases, leads to nonfunctional transgenes in the recipient genome. Parasitic plants are particularly prone to this phenomenon, but their mitochondrial genomes (mtDNA) have been largely unexplored. We undertook a large-scale mitochondrial genomic study of the holoparasitic plant Lophophytum mirabile (Balanophoraceae). Comprehensive phylogenetic analyses were performed to address the frequency, origin, and impact of HGT. The sequencing of the complete mtDNA of L. mirabile revealed the unprecedented acquisition of host-derived mitochondrial genes, representing 80% of the protein-coding gene content. All but two of these foreign genes replaced the native homologs and are probably functional in energy metabolism. The genome consists of 54 circular-mapping chromosomes, 25 of which carry no intact genes. The likely functional replacement of up to 26 genes in L. mirabile represents a stunning example of the potential effect of rampant HGT on plant mitochondria. The use of host-derived genes may have a positive effect on the host-parasite relationship, but could also be the result of nonadaptive forces.

  4. Gene deletion and allelic replacement in the filamentous fungus Podospora anserina.

    PubMed

    El-Khoury, Riyad; Sellem, Carole H; Coppin, Evelyne; Boivin, Antoine; Maas, Marc F P M; Debuchy, Robert; Sainsard-Chanet, Annie

    2008-04-01

    Gene replacement via homologous recombination is a fundamental tool for the analysis of gene function. However, this event is rare in organisms like the filamentous fungus Podospora anserina. We show here that deletion of the PaKu70 gene is an efficient strategy for improving gene manipulation in this organism. By using the DeltaPaKu70 strain, it is now possible (1) to produce deletion mutants with an efficiency of 100%, (2) to achieve allelic exchange by introducing a mutated allele associated with a selection cassette at the locus, (3) to introduce a mutation in a gene without co-insertion of a selectable marker and without any modification of the target locus.

  5. Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria

    PubMed Central

    Parikh, Amit; Kumar, Devanand; Chawla, Yogesh; Kurthkoti, Krishna; Khan, Shazia; Varshney, Umesh

    2013-01-01

    Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research. PMID:23315736

  6. Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

    PubMed

    Flisikowska, Tatiana; Thorey, Irmgard S; Offner, Sonja; Ros, Francesca; Lifke, Valeria; Zeitler, Bryan; Rottmann, Oswald; Vincent, Anna; Zhang, Lei; Jenkins, Shirin; Niersbach, Helmut; Kind, Alexander J; Gregory, Philip D; Schnieke, Angelika E; Platzer, Josef

    2011-01-01

    Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

  7. Bone marrow transplantation (BMT) and gene replacement therapy (GRT) in sickle cell anemia.

    PubMed

    Misaki, W

    2008-01-01

    Sickle cell anemia (SCA) forms one of the neglected tropical disease of genetic aetiology Unlike many complex genetic diseases inherited on a multiallelic pattern, SCA is a hemoglobinopathy of Mendelian type genetic inheritance. The SCA trait is inherited through a recessive autosomal link, with the homozygotes (SS) manifesting clinical disease, while the heterozygoste (AS) are clinically normal-exept in states of hypoxia or severe infection. Review of relevant literature on bone marrow transplantation and gene replacement therapy in sickle cell anaemia was obtained from texts and Pubmed search. The exact genetic disorder assailing SCA is the production of mutant or abnormal beta peptide chains in which the amino acid Glutamine has been substituted with valine. The issuing Haemoglobin polymerizes when deoxygenated,causing constituent RBCs to sickle, haemolyse and block small blood vessels- a clinical state that manifests as haemolytic anemia, aplasia, thrombo-embolic phenomenon, or painful crises. Unfortunately, up to day, there is no cure to SCA, and management only aims at preventing or ameliorating attacks. By virtue of its well defined and localized genetic aetiopathophysiology, and the advances in gene replacement therapy and regenerative medicine, we argue here the case for Bone marrow transplantation (BMT) and Gene replacement therapy (GRT) in sickle cell anemia.

  8. Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli.

    PubMed

    Herring, Christopher D; Glasner, Jeremy D; Blattner, Frederick R

    2003-06-05

    We have developed a method called "gene gorging" to make precise mutations in the Escherichia coli genome at frequencies high enough (1-15%) to allow direct identification of mutants by PCR or other screen rather than by selection. Gene gorging begins by establishing a donor plasmid carrying the desired mutation in the target cell. This plasmid is linearized by in vivo expression of the meganuclease I-SceI, providing a DNA substrate for lambda Red mediated recombination. This results in efficient replacement of the wild type allele on the chromosome with the modified sequence. We demonstrate gene gorging by introducing amber stop codons into the genes xylA, melA, galK, fucI, citA, ybdO, and lacZ. To compliment this approach we developed an arabinose inducible amber suppressor tRNA. Controlled expression mediated by the suppressor was demonstrated for the lacZ and xylA amber mutants.

  9. Identification of a GPER/GPR30 Antagonist with Improved Estrogen Receptor Counterselectivity

    PubMed Central

    Dennis, Megan K.; Field, Angela S.; Burai, Ritwik; Ramesh, Chinnasamy; Petrie, Whitney K.; Bologa, Cristian G.; Oprea, Tudor I.; Yamaguchi, Yuri; Hayashi, Shin-ichi; Sklar, S. Larry A.; Hathaway, Helen J.; Arterburn, Jeffrey B.; Prossnitz, Eric R.

    2011-01-01

    GPER/GPR30 is a seven-transmembrane G protein-coupled estrogen receptor that regulates many aspects of mammalian biology and physiology. We have previously described both a GPER-selective agonist G-1 and antagonist G15 based on a tetrahydro-3H-cyclopenta[c]quinoline scaffold. The antagonist lacks an ethanone moiety that likely forms important hydrogen bonds involved in receptor activation. Computational docking studies suggested that the lack of the ethanone substituent in G15 could minimize key steric conflicts, present in G-1, that limit binding within the ERα ligand binding pocket. In this report, we identify low-affinity cross-reactivity of the GPER antagonist G15 to the classical estrogen receptor ERα. To generate an antagonist with enhanced selectivity, we therefore synthesized an isosteric G-1 derivative, G36, containing an isopropyl moiety in place of the ethanone moiety. We demonstrate that G36 shows decreased binding and activation of ERα, while maintaining its antagonist profile towards GPER. G36 selectively inhibits estrogen-mediated activation of PI3K by GPER but not ERα. It also inhibits estrogen- and G-1-mediated calcium mobilization as well as ERK1/2 activation, with no effect on EGF-mediated ERK1/2 activation. Similar to G15, G36 inhibits estrogen- and G-1-stimulated proliferation of uterine epithelial cells in vivo. The identification of G36 as a GPER antagonist with improved ER counterselectivity represents a significant step towards the development of new highly selective therapeutics for cancer and other diseases. PMID:21782022

  10. Deletion of the BCSP31 gene of Brucella abortus by replacement.

    PubMed Central

    Halling, S M; Detilleux, P G; Tatum, F M; Judge, B A; Mayfield, J E

    1991-01-01

    The 31-kDa salt-extractable immunogenic protein, BCSP31, was deleted from several Brucella abortus strains by replacement with a marker gene encoding resistance to the antibiotics kanamycin and neomycin. The BCSP31 gene replacement plasmids, constructed with ColE1-derived vectors, were introduced by electroporation into B. abortus strain 19 (S19), into a rough variant of B. abortus S19, and into B. abortus S2308, and antibiotic-resistant transformants were isolated. B. abortus S19 is an attenuated strain used as a vaccine for prevention of bovine brucellosis in the United States, and B. abortus S2308 is a commonly used challenge strain. The antibiotic-resistant isolates were all obtained by recombination; none were spontaneous mutants. Loss of the gene encoding BCSP31 and presence of the marker gene were confirmed by Southern analysis. Vector sequences were either absent or linked to the genome, indicating that ColE1-derived plasmids are not maintained in B. abortus. Survival of B. abortus mutant strains in the macrophagelike cell line J774 and in HeLa cells was examined and shown to be indistinguishable from that of the parental strain. Images PMID:1937745

  11. Site-directed gene replacement of the phytopathogen Xanthomonas axonopodis pv. citri.

    PubMed

    Oshiro, Elisa E; Nepomuceno, Roberto S L; Faria, Juarez B; Ferreira, Luís C S; Ferreira, Rita C C

    2006-04-01

    In this work we defined experimental conditions for site-directed gene replacement of the Xanthomonas axonopodis pv. citri (Xac), an economically relevant pathogen of citrus plants. The procedure involved, first, optimizing the electrotransformation conditions of the Xac 306 strain and, second, constructing non-replicative suicide vectors carrying knockout copies of the target gene. Using specific experimental conditions, transformation efficiencies of Xac were at least 100 fold higher than those achieved with electroporation protocols previously designed for X. campestris transformation. Successful gene replacement events were achieved with a suicide vector derived from R6K plasmid (pWR-SS) but not with those with ColE1 replication origin. We have chosen the oppA as a target gene, encoding the binding component (OppA) of the major oligopeptide uptake system found in the genome of the Xac 306 strain, although not in X. campestris pv. campestris (Xcc). Defining the experimental conditions, which allow for the specific mutagenesis of the Xac 306 strain, represents a step in the understanding of both genetics and physiology of this economically important bacterial species.

  12. Natural replacement of vertically inherited lux-rib genes of Photobacterium aquimaris by horizontally acquired homologues.

    PubMed

    Urbanczyk, Henryk; Furukawa, Takashi; Yamamoto, Yuki; Dunlap, Paul V

    2012-08-01

    We report here the first instance of a complete replacement of vertically inherited luminescence genes by horizontally acquired homologues. Different strains of Photobacterium aquimaris contain homologues of the lux-rib genes that have a different evolutionary history. Strain BS1 from the Black Sea contains a vertically inherited lux-rib operon, which presumably arose in the ancestor of this species, whereas the type strain NBRC 104633(T) , from Sagami Bay, lacks the vertically inherited lux-rib operon and instead carries a complete and functional lux-rib operon acquired horizontally from a bacterium related to Photobacterium mandapamensis. The results indicate that the horizontal acquisition of the lux genes expanded the pan-genome of P. aquimaris, but it did not influence the phylogenetic divergence of this species.

  13. Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs.

    PubMed

    Kacar, Betül; Garmendia, Eva; Tuncbag, Nurcan; Andersson, Dan I; Hughes, Diarmaid

    2017-08-29

    Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufA gene in the Escherichia coli genome with its extant and ancestral homologs. The extant homologs represented tuf variants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 billion years ago (bya). Our results demonstrate that all of the foreign tuf genes are transferable to the E. coli genome, provided that an additional copy of the EF-Tu gene, tufB, remains present in the E. coli genome. However, when the tufB gene was removed, only the variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability of E. coli tuf with its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extant tuf homologs inserted into the E. coli genome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression of tuf partially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.IMPORTANCE Horizontal gene transfer (HGT) is a fundamental driving force in bacterial evolution. However, whether essential genes can be acquired by HGT and whether they can be acquired from distant organisms are very poorly understood. By systematically replacing tuf with ancestral homologs and homologs from

  14. SV40 vector with early gene replacement efficient in transducing exogenous DNA into mammalian cells.

    PubMed

    Asano, M; Iwakura, Y; Kawade, Y

    1985-12-09

    An early replacement SV40 vector, SV40-Mu beta, was constructed by replacing part of the early genes of the virus with mouse interferon-beta (IFN-beta) cDNA. Upon transfection of COS-7 cells with this DNA, transducing viral particles were produced, which could infect various cells and cause efficient production of mouse IFN-beta. The viral stock contained no detectable wild-type SV40. The IFN production after the virus infection was very high in monkey kidney cells, but less so in human epithelial cells, and low in mouse, pig, hamster cells and in human lymphocytes. The efficiency of introduction of the DNA to monkey kidney cells was compared with that by the calcium phosphate precipitation method, and the viral vector was found to be more efficient by a factor of several tens to hundreds.

  15. SYBR safe(TM) efficiently replaces ethidium bromide in Aspergillus fumigatus gene disruption.

    PubMed

    Canela, H M S; Takami, L A; Ferreira, M E S

    2017-02-08

    Invasive aspergillosis is a disease responsible for high mortality rates, caused mainly by Aspergillus fumigatus. The available drugs are limited and this disease continues to occur at an unacceptable frequency. Gene disruption is essential in the search for new drug targets. An efficient protocol for A. fumigatus gene disruption was described but it requires ethidium bromide, a genotoxic agent, for DNA staining. Therefore, the present study tested SYBR safe(TM), a non-genotoxic DNA stain, in A. fumigatus gene disruption protocol. The chosen gene was cipC, which has already been disrupted successfully in our laboratory. A deletion cassette was constructed in Saccharomyces cerevisiae and used in A. fumigatus transformation. There was no statistical difference between the tested DNA stains. The success rate of S. cerevisiae transformation was 63.3% for ethidium bromide and 70% for SYBR safe(TM). For A. fumigatus gene disruption, the success rate for ethidium bromide was 100 and 97% for SYBR safe(TM). In conclusion, SYBR safe(TM) efficiently replaced ethidium bromide, making this dye a safe and efficient alternative for DNA staining in A. fumigatus gene disruption.

  16. Inverse Medea as a novel gene drive system for local population replacement: a theoretical analysis.

    PubMed

    Marshall, John M; Hay, Bruce A

    2011-01-01

    One strategy to control mosquito-borne diseases, such as malaria and dengue fever, on a regional scale is to use gene drive systems to spread disease-refractory genes into wild mosquito populations. The development of a synthetic Medea element that has been shown to drive population replacement in laboratory Drosophila populations has provided encouragement for this strategy but has also been greeted with caution over the concern that transgenes may spread into countries without their consent. Here, we propose a novel gene drive system, inverse Medea, which is strong enough to bring about local population replacement but is unable to establish itself beyond an isolated release site. The system consists of 2 genetic components--a zygotic toxin and maternal antidote--which render heterozygous offspring of wild-type mothers unviable. Through population genetic analysis, we show that inverse Medea will only spread when it represents a majority of the alleles in a population. The element is best located on an autosome and will spread to fixation provided any associated fitness costs are dominant and to very high frequency otherwise. We suggest molecular tools that could be used to build the inverse Medea system and discuss its utility for a confined release of transgenic mosquitoes.

  17. Inverse Medea as a Novel Gene Drive System for Local Population Replacement: A Theoretical Analysis

    PubMed Central

    Hay, Bruce A.

    2011-01-01

    One strategy to control mosquito-borne diseases, such as malaria and dengue fever, on a regional scale is to use gene drive systems to spread disease-refractory genes into wild mosquito populations. The development of a synthetic Medea element that has been shown to drive population replacement in laboratory Drosophila populations has provided encouragement for this strategy but has also been greeted with caution over the concern that transgenes may spread into countries without their consent. Here, we propose a novel gene drive system, inverse Medea, which is strong enough to bring about local population replacement but is unable to establish itself beyond an isolated release site. The system consists of 2 genetic components—a zygotic toxin and maternal antidote—which render heterozygous offspring of wild-type mothers unviable. Through population genetic analysis, we show that inverse Medea will only spread when it represents a majority of the alleles in a population. The element is best located on an autosome and will spread to fixation provided any associated fitness costs are dominant and to very high frequency otherwise. We suggest molecular tools that could be used to build the inverse Medea system and discuss its utility for a confined release of transgenic mosquitoes. PMID:21493596

  18. The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation

    PubMed Central

    Kwiatek, Agnieszka; Bacal, Pawel; Wasiluk, Adrian; Trybunko, Anastasiya; Adamczyk-Poplawska, Monika

    2014-01-01

    Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than

  19. The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation.

    PubMed

    Kwiatek, Agnieszka; Bacal, Pawel; Wasiluk, Adrian; Trybunko, Anastasiya; Adamczyk-Poplawska, Monika

    2014-01-01

    Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than

  20. CRISPR/Cas9 and mitochondrial gene replacement therapy: promising techniques and ethical considerations

    PubMed Central

    Fogleman, Sarah; Santana, Casey; Bishop, Casey; Miller, Alyssa; Capco, David G

    2016-01-01

    Thousands of mothers are at risk of transmitting mitochondrial diseases to their offspring each year, with the most severe form of these diseases being fatal [1]. With no cure, transmission prevention is the only current hope for decreasing the disease incidence. Current methods of prevention rely on low mutant maternal mitochondrial DNA levels, while those with levels close to or above threshold (>60%) are still at a very high risk of transmission [2]. Two novel approaches may offer hope for preventing and treating mitochondrial disease: mitochondrial replacement therapy, and CRISPR/Cas9. Mitochondrial replacement therapy has emerged as a promising tool that has the potential to prevent transmission in patients with higher mutant mitochondrial loads. This method is the subject of many ethical concerns due its use of a donor embryo to transplant the patient’s nuclear DNA; however, it has ultimately been approved for use in the United Kingdom and was recently declared ethically permissible by the FDA. The leading-edge CRISPR/Cas9 technology exploits the principles of bacterial immune function to target and remove specific sequences of mutated DNA. This may have potential in treating individuals with disease caused by mutant mitochondrial DNA. As the technology progresses, it is important that the ethical considerations herein emerge and become more established. The purpose of this review is to discuss current research surrounding the procedure and efficacy of the techniques, compare the ethical concerns of each approach, and look into the future of mitochondrial gene replacement therapy. PMID:27725916

  1. Engineered cellular gene-replacement platform for selective and inducible proteolytic profiling

    PubMed Central

    Morgan, Charles W.; Diaz, Juan E.; Zeitlin, Samantha G.; Gray, Daniel C.; Wells, James A.

    2015-01-01

    Cellular demolition during apoptosis is completed by executioner caspases, that selectively cleave more than 1,500 proteins but whose individual roles are challenging to assess. Here, we used an optimized site-specific and inducible protease to examine the role of a classic apoptotic node, the caspase-activated DNase (CAD). CAD is activated when caspases cleave its endogenous inhibitor ICAD, resulting in the characteristic DNA laddering of apoptosis. We describe a posttranscriptional gene replacement (PTGR) approach where endogenous biallelic ICAD is knocked down and simultaneously replaced with an engineered allele that is susceptible to inducible cleavage by tobacco etch virus protease. Remarkably, selective activation of CAD alone does not induce cell death, although hallmarks of DNA damage are detected in human cancer cell lines. Our data strongly support that the highly cooperative action of CAD and inhibition of DNA repair systems are critical for the DNA laddering phenotype in apoptosis. Furthermore, the PTGR approach provides a general means for replacing wild-type protein function with a precisely engineered mutant at the transcriptional level that should be useful for cell engineering studies. PMID:26106156

  2. CRISPR/Cas9 and mitochondrial gene replacement therapy: promising techniques and ethical considerations.

    PubMed

    Fogleman, Sarah; Santana, Casey; Bishop, Casey; Miller, Alyssa; Capco, David G

    2016-01-01

    Thousands of mothers are at risk of transmitting mitochondrial diseases to their offspring each year, with the most severe form of these diseases being fatal [1]. With no cure, transmission prevention is the only current hope for decreasing the disease incidence. Current methods of prevention rely on low mutant maternal mitochondrial DNA levels, while those with levels close to or above threshold (>60%) are still at a very high risk of transmission [2]. Two novel approaches may offer hope for preventing and treating mitochondrial disease: mitochondrial replacement therapy, and CRISPR/Cas9. Mitochondrial replacement therapy has emerged as a promising tool that has the potential to prevent transmission in patients with higher mutant mitochondrial loads. This method is the subject of many ethical concerns due its use of a donor embryo to transplant the patient's nuclear DNA; however, it has ultimately been approved for use in the United Kingdom and was recently declared ethically permissible by the FDA. The leading-edge CRISPR/Cas9 technology exploits the principles of bacterial immune function to target and remove specific sequences of mutated DNA. This may have potential in treating individuals with disease caused by mutant mitochondrial DNA. As the technology progresses, it is important that the ethical considerations herein emerge and become more established. The purpose of this review is to discuss current research surrounding the procedure and efficacy of the techniques, compare the ethical concerns of each approach, and look into the future of mitochondrial gene replacement therapy.

  3. MEN1 gene replacement therapy reduces proliferation rates in a mouse model of pituitary adenomas.

    PubMed

    Walls, Gerard V; Lemos, Manuel C; Javid, Mahsa; Bazan-Peregrino, Miriam; Jeyabalan, Jeshmi; Reed, Anita A C; Harding, Brian; Tyler, Damian J; Stuckey, Daniel J; Piret, Sian; Christie, Paul T; Ansorge, Olaf; Clarke, Kieran; Seymour, Len; Thakker, Rajesh V

    2012-10-01

    Multiple endocrine neoplasia type 1 (MEN1) is characterized by the combined occurrence of pituitary, pancreatic, and parathyroid tumors showing loss of heterozygosity in the putative tumor suppressor gene MEN1. This gene encodes the protein menin, the overexpression of which inhibits cell proliferation in vitro. In this study, we conducted a preclinical evaluation of MEN1 gene therapy in pituitary tumors of Men1(+/-) mice, using a recombinant nonreplicating adenoviral serotype 5 vector that contained the murine Men1 cDNA under control of a cytomegalovirus promoter (Men1.rAd5). Pituitary tumors in 55 Men1(+/-) female mice received a transauricular intratumoral injection of Men1.rAd5 or control treatments, followed by 5-bromo-2-deoxyuridine (BrdUrd) in drinking water for four weeks before magnetic resonance imaging (MRI) and immunohistochemical analysis. Immediate procedure-related and 4-week mortalities were similar in all groups, indicating that the adenoviral gene therapy was not associated with a higher mortality. Menin expression was higher in the Men1.rAd5-treated mice when compared with other groups. Daily proliferation rates assessed by BrdUrd incorporation were reduced significantly in Men1.rAd5-injected tumors relative to control-treated tumors. In contrast, apoptotic rates, immune T-cell response, and tumor volumes remained similar in all groups. Our findings establish that MEN1 gene replacement therapy can generate menin expression in pituitary tumors, and significantly reduce tumor cell proliferation. ©2012 AACR.

  4. Potential impact of human mitochondrial replacement on global policy regarding germline gene modification.

    PubMed

    Ishii, Tetsuya

    2014-08-01

    Previous discussions regarding human germline gene modification led to a global consensus that no germline should undergo genetic modification. However, the UK Human Fertilisation and Embryology Authority, having conducted at the UK Government's request a scientific review and a wide public consultation, provided advice to the Government on the pros and cons of Parliament's lifting a ban on altering mitochondrial DNA content of human oocytes and embryos, so as to permit the prevention of maternal transmission of mitochondrial diseases. In this commentary, relevant ethical and biomedical issues are examined and requirements for proceeding with this novel procedure are suggested. Additionally, potentially significant impacts of the UK legalization on global policy concerning germline gene modification are discussed in the context of recent advances in genome-editing technology. It is concluded that international harmonization is needed, as well as further ethical and practical consideration, prior to the legalization of human mitochondrial replacement.

  5. Gene Replacement for the Generation of Designed Novel Avermectin Derivatives with Enhanced Acaricidal and Nematicidal Activities

    PubMed Central

    Huang, Jun; Chen, An-Liang; Zhang, Hui; Yu, Zhen; Li, Mei-Hong; Li, Na; Lin, Jia-Tan; Bai, Hua

    2015-01-01

    Avermectin (AVM) and ivermectin (IVM) are potent pesticides and acaricides which have been widely used during the past 30 years. As insect resistance to AVM and IVM is greatly increasing, alternatives are urgently needed. Here, we report two novel AVM derivatives, tenvermectin A (TVM A) and TVM B, which are considered a potential new generation of agricultural and veterinary drugs. The molecules of the TVMs were designed based on structure and pharmacological property comparisons among AVM, IVM, and milbemycin (MBM). To produce TVMs, a genetically engineered strain, MHJ1011, was constructed from Streptomyces avermitilis G8-17, an AVM industrial strain. In MHJ1011, the native aveA1 gene was seamlessly replaced with milA1 from Streptomyces hygroscopicus. The total titer of the two TVMs produced by MHJ1011 reached 3,400 mg/liter. Insecticidal tests proved that TVM had enhanced activities against Tetranychus cinnabarinus and Bursaphelenchus xylophilus, as desired. This study provides a typical example of exploration for novel active compounds through a new method of polyketide synthase (PKS) reassembly for gene replacement. The results of the insecticidal tests may be of use in elucidating the structure-activity relationship of AVMs and MBMs. PMID:26025902

  6. Gene Replacement for the Generation of Designed Novel Avermectin Derivatives with Enhanced Acaricidal and Nematicidal Activities.

    PubMed

    Huang, Jun; Chen, An-Liang; Zhang, Hui; Yu, Zhen; Li, Mei-Hong; Li, Na; Lin, Jia-Tan; Bai, Hua; Wang, Ji-Dong; Zheng, Yu-Guo

    2015-08-15

    Avermectin (AVM) and ivermectin (IVM) are potent pesticides and acaricides which have been widely used during the past 30 years. As insect resistance to AVM and IVM is greatly increasing, alternatives are urgently needed. Here, we report two novel AVM derivatives, tenvermectin A (TVM A) and TVM B, which are considered a potential new generation of agricultural and veterinary drugs. The molecules of the TVMs were designed based on structure and pharmacological property comparisons among AVM, IVM, and milbemycin (MBM). To produce TVMs, a genetically engineered strain, MHJ1011, was constructed from Streptomyces avermitilis G8-17, an AVM industrial strain. In MHJ1011, the native aveA1 gene was seamlessly replaced with milA1 from Streptomyces hygroscopicus. The total titer of the two TVMs produced by MHJ1011 reached 3,400 mg/liter. Insecticidal tests proved that TVM had enhanced activities against Tetranychus cinnabarinus and Bursaphelenchus xylophilus, as desired. This study provides a typical example of exploration for novel active compounds through a new method of polyketide synthase (PKS) reassembly for gene replacement. The results of the insecticidal tests may be of use in elucidating the structure-activity relationship of AVMs and MBMs.

  7. An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design.

    PubMed

    Zhao, Yongping; Zhang, Congsheng; Liu, Wenwen; Gao, Wei; Liu, Changlin; Song, Gaoyuan; Li, Wen-Xue; Mao, Long; Chen, Beijiu; Xu, Yunbi; Li, Xinhai; Xie, Chuanxiao

    2016-04-01

    Precision DNA/gene replacement is a promising genome-editing tool that is highly desirable for molecular engineering and breeding by design. Although the CRISPR/Cas9 system works well as a tool for gene knockout in plants, gene replacement has rarely been reported. Towards this end, we first designed a combinatory dual-sgRNA/Cas9 vector (construct #1) that successfully deleted miRNA gene regions (MIR169a and MIR827a). The deletions were confirmed by PCR and subsequent sequencing, yielding deletion efficiencies of 20% and 24% on MIR169a and MIR827a loci, respectively. We designed a second structure (construct #2) that contains sites homologous to Arabidopsis TERMINAL FLOWER 1 (TFL1) for homology-directed repair (HDR) with regions corresponding to the two sgRNAs on the modified construct #1. The two constructs were co-transformed into Arabidopsis plants to provide both targeted deletion and donor repair for targeted gene replacement by HDR. Four of 500 stably transformed T0 transgenic plants (0.8%) contained replaced fragments. The presence of the expected recombination sites was further confirmed by sequencing. Therefore, we successfully established a gene deletion/replacement system in stably transformed plants that can potentially be utilized to introduce genes of interest for targeted crop improvement.

  8. Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs

    PubMed Central

    Garmendia, Eva; Tuncbag, Nurcan; Andersson, Dan I.; Hughes, Diarmaid

    2017-01-01

    ABSTRACT Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufA gene in the Escherichia coli genome with its extant and ancestral homologs. The extant homologs represented tuf variants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 billion years ago (bya). Our results demonstrate that all of the foreign tuf genes are transferable to the E. coli genome, provided that an additional copy of the EF-Tu gene, tufB, remains present in the E. coli genome. However, when the tufB gene was removed, only the variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability of E. coli tuf with its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extant tuf homologs inserted into the E. coli genome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression of tuf partially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria. PMID:28851849

  9. Excretion of Antibiotic Resistance Genes by Dairy Calves Fed Milk Replacers with Varying Doses of Antibiotics

    PubMed Central

    Thames, Callie H.; Pruden, Amy; James, Robert E.; Ray, Partha P.; Knowlton, Katharine F.

    2012-01-01

    Elevated levels of antibiotic resistance genes (ARGs) in soil and water have been linked to livestock farms and in some cases feed antibiotics may select for antibiotic resistant gut microbiota. The purpose of this study was to examine the establishment of ARGs in the feces of calves receiving milk replacer containing no antibiotics versus subtherapeutic or therapeutic doses of tetracycline and neomycin. The effect of antibiotics on calf health was also of interest. Twenty-eight male and female dairy calves were assigned to one of the three antibiotic treatment groups at birth and fecal samples were collected at weeks 6, 7 (prior to weaning), and 12 (5 weeks after weaning). ARGs corresponding to the tetracycline (tetC, tetG, tetO, tetW, and tetX), macrolide (ermB, ermF), and sulfonamide (sul1, sul2) classes of antibiotics along with the class I integron gene, intI1, were monitored by quantitative polymerase chain reaction as potential indicators of direct selection, co-selection, or horizontal gene transfer of ARGs. Surprisingly, there was no significant effect of antibiotic treatment on the absolute abundance (gene copies per gram wet manure) of any of the ARGs except ermF, which was lower in the antibiotic-treated calf manure, presumably because a significant portion of host bacterial cells carrying ermF were not resistant to tetracycline or neomycin. However, relative abundance (gene copies normalized to 16S rRNA genes) of tetO was higher in calves fed the highest dose of antibiotic than in the other treatments. All genes, except tetC and intI1, were detectable in feces from 6 weeks onward, and tetW and tetG significantly increased (P < 0.10), even in control calves. Overall, the results provide new insight into the colonization of calf gut flora with ARGs in the early weeks. Although feed antibiotics exerted little effect on the ARGs monitored in this study, the fact that they also provided no health benefit suggests that the greater than conventional

  10. Topical cystic fibrosis transmembrane conductance regulator gene replacement for cystic fibrosis-related lung disease.

    PubMed

    Lee, Tim W R; Southern, Kevin W; Perry, Luke A; Penny-Dimri, Jahan C; Aslam, Aisha A

    2016-06-17

    Cystic fibrosis is caused by a defective gene encoding a protein called the cystic fibrosis transmembrane conductance regulator (CFTR), and is characterised by chronic lung infection resulting in inflammation and progressive lung damage that results in a reduced life expectancy. To determine whether topical CFTR gene replacement therapy to the lungs in people with cystic fibrosis is associated with improvements in clinical outcomes, and to assess any adverse effects. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches, handsearching relevant journals and abstract books of conference proceedings.Date of most recent search: 05 May 2016.An additional search of the National Institutes for Health (NIH) Genetic Modification Clinical Research Information System (GeMCRIS) was also performed for the years 1992 to 2015.Date of most recent search: 20 April 2016. Randomised controlled studies comparing topical CFTR gene delivery to the lung, using either viral or non-viral delivery systems, with placebo or an alternative delivery system in people with confirmed cystic fibrosis. The authors independently extracted data and assessed study quality. Authors of included studies were contacted and asked for any available additional data. Meta-analysis was limited due to differing study designs. Four randomised controlled studies met the inclusion criteria for this review, involving a total of 302 participants lasting from 29 days to 13 months; 14 studies were excluded. The included studies differed in terms of CFTR gene replacement agent and study design, which limited the meta-analysis. One study only enrolled adult males, the remaining studies included both males and females aged 12 years and over.Risk of bias in the studies was moderate. Random sequence generation and allocation concealment was only described in the more recent study; the remaining three studies were

  11. Inhibition of RecBCD in Klebsiella pneumoniae by Gam and its effect on the efficiency of gene replacement.

    PubMed

    Chen, Chuan; Wei, Dong; Liu, Pengfu; Wang, Min; Shi, Jiping; Jiang, Biao; Hao, Jian

    2016-02-01

    Gam protein is an inhibitor of the host RecBCD exonuclease, and this inhibition is essential to the proficiency of Red recombinase-mediated gene replacement. In Klebsiella pneumoniae, the efficiency of this gene replacement was lower than that in Escherichia coli, and the minimum length of homologous extensions required was longer. Thus, it was supposed that the inhibitory effect of Gam against RecBCD was weak in K. pneumoniae. To test this hypothesis, a Gam-deficient Red recombinase expression plasmid and a ΔrecB K. pneumoniae mutant were constructed. The Gam-deficient Red recombinase showed a reduced capacity for gene replacement compared with that of the complete Red recombinase. The efficiency of gene replacement in the ΔrecB mutant was 6-8 times higher than the wild-type strain, and the minimum length for the homologous extensions was reduced to 100 bp. These results indicate that Gam does inhibit the RecBCD exonuclease in K. pneumoniae, but that this inhibition is not stringent. Furthermore, mutation of recB presents a convenient and efficient method to enhance the Red recombinase assisted gene replacement in K. pneumoniae.

  12. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes.

    PubMed

    Flax, J D; Aurora, S; Yang, C; Simonin, C; Wills, A M; Billinghurst, L L; Jendoubi, M; Sidman, R L; Wolfe, J H; Kim, S U; Snyder, E Y

    1998-11-01

    Stable clones of neural stem cells (NSCs) have been isolated from the human fetal telencephalon. These self-renewing clones give rise to all fundamental neural lineages in vitro. Following transplantation into germinal zones of the newborn mouse brain they participate in aspects of normal development, including migration along established migratory pathways to disseminated central nervous system regions, differentiation into multiple developmentally and regionally appropriate cell types, and nondisruptive interspersion with host progenitors and their progeny. These human NSCs can be genetically engineered and are capable of expressing foreign transgenes in vivo. Supporting their gene therapy potential, secretory products from NSCs can correct a prototypical genetic metabolic defect in neurons and glia in vitro. The human NSCs can also replace specific deficient neuronal populations. Cryopreservable human NSCs may be propagated by both epigenetic and genetic means that are comparably safe and effective. By analogy to rodent NSCs, these observations may allow the development of NSC transplantation for a range of disorders.

  13. Successful RPE65 gene replacement and improved visual function in humans.

    PubMed

    Koenekoop, Robert K

    2008-09-01

    Leber congenital amaurosis (LCA) is a group of severe autosomal recessive retinal dystrophies unified by the onset of blindness at birth and absence of ERG signals. Mutations in fourteen genes are currently associated with LCA, accounting for approximately 60% of patients. LCA genes encode retinal proteins that participate in a variety of significant retinal pathways ranging from photoreceptor development to replenishing vitamin A in the retinoid cycle. In some of the genetic subtypes of LCA (e.g. RPE65), viable photoreceptors and a relatively intact retina have been discovered. Consequently, successful photoreceptor and visual rescue have been achieved in mice and canine models of LCA. Two research groups, one in Philadelphia, USA (Maguire et al.) another in London, England (Bainbridge et al.) recently tested RPE65 replacement in two human trials with a total of six LCA patients. Remarkably, visual improvements were documented by ETDRS visual acuity measurements, pupillometry, nystagmus frequency, visual field measures, perimetry and an obstacle course. There were no local, or systemic side effects, nor improvements in ERG measures. These spectacular results will now stimulate further investigations of younger patients, higher dosages, and clinically or genetically related retinal disorders.

  14. Mitochondrial gene replacement in human pluripotent stem cell-derived neural progenitors.

    PubMed

    Iyer, S; Xiao, E; Alsayegh, K; Eroshenko, N; Riggs, M J; Bennett, J P; Rao, R R

    2012-05-01

    Human pluripotent stem cell-derived neural progenitor (hNP) cells are an excellent resource for understanding early neural development and neurodegenerative disorders. Given that many neurodegenerative disorders can be correlated with defects in the mitochondrial genome, optimal utilization of hNP cells requires an ability to manipulate and monitor changes in the mitochondria. Here, we describe a novel approach that uses recombinant human mitochondrial transcription factor A (rhTFAM) protein to transfect and express a pathogenic mitochondrial genome (mtDNA) carrying the G11778A mutation associated with Leber's hereditary optic neuropathy (LHON) disease, into dideoxycytidine (ddC)-treated hNPs. Treatment with ddC reduced endogenous mtDNA and gene expression, without loss of hNP phenotypic markers. Entry of G11778A mtDNA complexed with the rhTFAM was observed in mitochondria of ddC-hNPs. Expression of the pathogenic RNA was confirmed by restriction enzyme analysis of the SfaN1-digested cDNA. On the basis of the expression of neuron-specific class III beta-tubulin, neuronal differentiation occurred. Our results show for the first time that pathogenic mtDNA can be introduced and expressed into hNPs without loss of phenotype or neuronal differentiation potential. This mitochondrial gene replacement technology allows for creation of in vitro stem cell-based models useful for understanding neuronal development and treatment of neurodegenerative disorders.

  15. Editing Transgenic DNA Components by Inducible Gene Replacement in Drosophila melanogaster

    PubMed Central

    Lin, Chun-Chieh; Potter, Christopher J.

    2016-01-01

    Gene conversions occur when genomic double-strand DNA breaks (DSBs) trigger unidirectional transfer of genetic material from a homologous template sequence. Exogenous or mutated sequence can be introduced through this homology-directed repair (HDR). We leveraged gene conversion to develop a method for genomic editing of existing transgenic insertions in Drosophila melanogaster. The clustered regularly-interspaced palindromic repeats (CRISPR)/Cas9 system is used in the homology assisted CRISPR knock-in (HACK) method to induce DSBs in a GAL4 transgene, which is repaired by a single-genomic transgenic construct containing GAL4 homologous sequences flanking a T2A-QF2 cassette. With two crosses, this technique converts existing GAL4 lines, including enhancer traps, into functional QF2 expressing lines. We used HACK to convert the most commonly-used GAL4 lines (labeling tissues such as neurons, fat, glia, muscle, and hemocytes) to QF2 lines. We also identified regions of the genome that exhibited differential efficiencies of HDR. The HACK technique is robust and readily adaptable for targeting and replacement of other genomic sequences, and could be a useful approach to repurpose existing transgenes as new genetic reagents become available. PMID:27334272

  16. Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.

    PubMed Central

    Pyne, C; Bognar, A L

    1992-01-01

    The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products. Images PMID:1548226

  17. Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum.

    PubMed

    Gilles, Anna F; Schinko, Johannes B; Averof, Michalis

    2015-08-15

    Gene-editing techniques are revolutionizing the way we conduct genetics in many organisms. The CRISPR/Cas nuclease has emerged as a highly versatile, efficient and affordable tool for targeting chosen sites in the genome. Beyond its applications in established model organisms, CRISPR technology provides a platform for genetic intervention in a wide range of species, limited only by our ability to deliver it to cells and to select mutations efficiently. Here, we test the CRISPR technology in an emerging insect model and pest, the beetle Tribolium castaneum. We use simple assays to test CRISPR/Cas activity, we demonstrate efficient expression of guide RNAs and Cas9 from Tribolium U6 and hsp68 promoters and we test the efficiency of knockout and knock-in approaches in Tribolium. We find that 55-80% of injected individuals carry mutations (indels) generated by non-homologous end joining, including mosaic bi-allelic knockouts; 71-100% carry such mutations in their germ line and transmit them to the next generation. We show that CRISPR-mediated gene knockout of the Tribolium E-cadherin gene causes defects in dorsal closure, which is consistent with RNAi-induced phenotypes. Homology-directed knock-in of marker transgenes was observed in 14% of injected individuals and transmitted to the next generation by 6% of injected individuals. Previous work in Tribolium mapped a large number of transgene insertions associated with developmental phenotypes and enhancer traps. We present an efficient method for re-purposing these insertions, via CRISPR-mediated replacement of these transgenes by new constructs.

  18. Replacement Gene Therapy with a Human RPGRIP1 Sequence Slows Photoreceptor Degeneration in a Murine Model of Leber Congenital Amaurosis

    PubMed Central

    Pawlyk, Basil S.; Bulgakov, Oleg V.; Liu, Xiaoqing; Xu, Xiaoyun; Adamian, Michael; Sun, Xun; Khani, Shahrokh C.; Berson, Eliot L.; Sandberg, Michael A.

    2010-01-01

    Abstract RPGR-interacting protein-1 (RPGRIP1) is localized in the photoreceptor-connecting cilium, where it anchors the RPGR (retinitis pigmentosa GTPase regulator) protein, and its function is essential for photoreceptor maintenance. Genetic defect in RPGRIP1 is a known cause of Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. We evaluated the efficacy of replacement gene therapy in a murine model of LCA carrying a targeted disruption of RPGRIP1. The replacement construct, packaged in an adeno-associated virus serotype 8 (AAV8) vector, used a rhodopsin kinase gene promoter to drive RPGRIP1 expression. Both promoter and transgene were of human origin. After subretinal delivery of the replacement gene in the mutant mice, human RPGRIP1 was expressed specifically in photoreceptors, localized correctly in the connecting cilia, and restored the normal localization of RPGR. Electroretinogram and histological examinations showed better preservation of rod and cone photoreceptor function and improved photoreceptor survival in the treated eyes. This study demonstrates the efficacy of human gene replacement therapy and validates a gene therapy design for future clinical trials in patients afflicted with this condition. Our results also have therapeutic implications for other forms of retinal degenerations attributable to a ciliary defect. PMID:20384479

  19. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin.

    PubMed

    Gust, Bertolt; Challis, Greg L; Fowler, Kay; Kieser, Tobias; Chater, Keith F

    2003-02-18

    Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and lambda-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriT(RK2) for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT(RK2) to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.

  20. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

    PubMed Central

    Gust, Bertolt; Challis, Greg L.; Fowler, Kay; Kieser, Tobias; Chater, Keith F.

    2003-01-01

    Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and λ-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriTRK2 for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriTRK2 to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate. PMID:12563033

  1. A long-term efficacy study of gene replacement therapy for RPGR-associated retinal degeneration

    PubMed Central

    Wu, Zhijian; Hiriyanna, Suja; Qian, Haohua; Mookherjee, Suddhasil; Campos, Maria M.; Gao, Chun; Fariss, Robert; Sieving, Paul A.; Li, Tiansen; Colosi, Peter; Swaroop, Anand

    2015-01-01

    Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for >70% of X-linked retinitis pigmentosa (XLRP) and 15–20% of all inherited retinal degeneration. Gene replacement therapy for RPGR-XLRP was hampered by the relatively slow disease progression in mouse models and by difficulties in cloning the full-length RPGR-ORF15 cDNA that includes a purine-rich 3′-coding region; however, its effectiveness has recently been demonstrated in four dogs with RPGR mutations. To advance the therapy to clinical stage, we generated new stable vectors in AAV8 or AAV9 carrying mouse and human full-length RPGR-ORF15-coding sequence and conducted a comprehensive long-term dose-efficacy study in Rpgr-knockout mice. After validating their ability to produce full-length proteins that localize to photoreceptor connecting cilia, we evaluated various vector doses in mice during a 2-year study. We demonstrate that eyes treated with a single injection of mouse or human RPGR-ORF15 vector at an optimal dose maintained the expression of RPGR-ORF15 throughout the study duration and exhibited higher electroretinogram amplitude, thicker photoreceptor layer and better targeting of opsins to outer segments compared with sham-treated eyes. Furthermore, mice that received treatment at an advanced age also showed remarkable preservation of retinal structure and function. Retinal toxicity was observed at high vector doses, highlighting the importance of careful dose optimization in future clinical experiments. Our long-term dose-efficacy study should facilitate the design of human trials with human RPGR-ORF15 vector as a clinical candidate. PMID:25877300

  2. Antipathogen genes and the replacement of disease-vectoring mosquito populations: a model-based evaluation

    PubMed Central

    Robert, Michael A; Okamoto, Kenichi W; Gould, Fred; Lloyd, Alun L

    2014-01-01

    Recently, genetic strategies aimed at controlling populations of disease-vectoring mosquitoes have received considerable attention as alternatives to traditional measures. Theoretical studies have shown that female-killing (FK), antipathogen (AP), and reduce and replace (R&R) strategies can each decrease the number competent vectors. In this study, we utilize a mathematical model to evaluate impacts on competent Aedes aegypti populations of FK, AP, and R&R releases as well as hybrid strategies that result from combinations of these three approaches. We show that while the ordering of efficacy of these strategies depends upon population life history parameters, sex ratio of releases, and switch time in combination strategies, AP-only and R&R/AP releases typically lead to the greatest long-term reduction in competent vectors. R&R-only releases are often less effective at long-term reduction of competent vectors than AP-only releases or R&R/AP releases. Furthermore, the reduction in competent vectors caused by AP-only releases is easier to maintain than that caused by FK-only or R&R-only releases even when the AP gene confers a fitness cost. We discuss the roles that density dependence and inclusion of females play in the order of efficacy of the strategies. We anticipate that our results will provide added impetus to continue developing AP strategies. PMID:25558284

  3. Evaluation of MYBPC3 trans-Splicing and Gene Replacement as Therapeutic Options in Human iPSC-Derived Cardiomyocytes.

    PubMed

    Prondzynski, Maksymilian; Krämer, Elisabeth; Laufer, Sandra D; Shibamiya, Aya; Pless, Ole; Flenner, Frederik; Müller, Oliver J; Münch, Julia; Redwood, Charles; Hansen, Arne; Patten, Monica; Eschenhagen, Thomas; Mearini, Giulia; Carrier, Lucie

    2017-06-16

    Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM) carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ∼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5' or 3' pre-trans-splicing molecules represented ∼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ∼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1) the feasibility of trans-splicing, although with low efficiency, and (2) efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  4. Establishment of a Counter-selectable Markerless Mutagenesis System in Veillonella atypica

    PubMed Central

    Zhou, Peng; Li, Xiaoli; Qi, Fengxia

    2015-01-01

    Using an alternative sigma factor ecf3 as target, we successfully established the first markerless mutagenesis system in the Veillonella genus. This system will be a valuable tool for mutagenesis of multiple genes for gene function analysis as well as for gene regulation studies in Veillonella. PMID:25771833

  5. Accumulation of VH Replacement Products in IgH Genes Derived from Autoimmune Diseases and Anti-Viral Responses in Human

    PubMed Central

    Lange, Miles D.; Huang, Lin; Yu, Yangsheng; Li, Song; Liao, Hongyan; Zemlin, Michael; Su, Kaihong; Zhang, Zhixin

    2014-01-01

    VH replacement refers to RAG-mediated secondary recombination of the IgH genes, which renews almost the entire VH gene coding region but retains a short stretch of nucleotides as a VH replacement footprint at the newly generated VH–DH junction. To explore the biological significance of VH replacement to the antibody repertoire, we developed a Java-based VH replacement footprint analyzer program and analyzed the distribution of VH replacement products in 61,851 human IgH gene sequences downloaded from the NCBI database. The initial assignment of the VH, DH, and JH gene segments provided a comprehensive view of the human IgH repertoire. To our interest, the overall frequency of VH replacement products is 12.1%; the frequencies of VH replacement products in IgH genes using different VH germline genes vary significantly. Importantly, the frequencies of VH replacement products are significantly elevated in IgH genes derived from different autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and allergic rhinitis, and in IgH genes encoding various autoantibodies or anti-viral antibodies. The identified VH replacement footprints preferentially encoded charged amino acids to elongate IgH CDR3 regions, which may contribute to their autoreactivities or anti-viral functions. Analyses of the mutation status of the identified VH replacement products suggested that they had been actively involved in immune responses. These results provide a global view of the distribution of VH replacement products in human IgH genes, especially in IgH genes derived from autoimmune diseases and anti-viral immune responses. PMID:25101087

  6. Accumulation of VH Replacement Products in IgH Genes Derived from Autoimmune Diseases and Anti-Viral Responses in Human.

    PubMed

    Lange, Miles D; Huang, Lin; Yu, Yangsheng; Li, Song; Liao, Hongyan; Zemlin, Michael; Su, Kaihong; Zhang, Zhixin

    2014-01-01

    VH replacement refers to RAG-mediated secondary recombination of the IgH genes, which renews almost the entire VH gene coding region but retains a short stretch of nucleotides as a VH replacement footprint at the newly generated VH-DH junction. To explore the biological significance of VH replacement to the antibody repertoire, we developed a Java-based VH replacement footprint analyzer program and analyzed the distribution of VH replacement products in 61,851 human IgH gene sequences downloaded from the NCBI database. The initial assignment of the VH, DH, and JH gene segments provided a comprehensive view of the human IgH repertoire. To our interest, the overall frequency of VH replacement products is 12.1%; the frequencies of VH replacement products in IgH genes using different VH germline genes vary significantly. Importantly, the frequencies of VH replacement products are significantly elevated in IgH genes derived from different autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and allergic rhinitis, and in IgH genes encoding various autoantibodies or anti-viral antibodies. The identified VH replacement footprints preferentially encoded charged amino acids to elongate IgH CDR3 regions, which may contribute to their autoreactivities or anti-viral functions. Analyses of the mutation status of the identified VH replacement products suggested that they had been actively involved in immune responses. These results provide a global view of the distribution of VH replacement products in human IgH genes, especially in IgH genes derived from autoimmune diseases and anti-viral immune responses.

  7. CRISPR/Cas9-induced Targeted Mutagenesis and Gene Replacement to Generate Long-shelf Life Tomato Lines.

    PubMed

    Yu, Qing-Hui; Wang, Baike; Li, Ning; Tang, Yaping; Yang, Shengbao; Yang, Tao; Xu, Juan; Guo, Chunmiao; Yan, Peng; Wang, Qiang; Asmutola, Patiguli

    2017-09-19

    Quickly and precisely gain genetically enhanced breeding elites with value-adding performance traits is desired by the crop breeders all the time. The present of gene editing technologies, especially the CRISPR/Cas9 system with the capacities of efficiency, versatility and multiplexing provides a reasonable expectation towards breeding goals. For exploiting possible application to accelerate the speed of process at breeding by CRISPR/Cas9 technology, in this study, the Agrobacterium tumefaciens-mediated CRISPR/Cas9 system transformation method was used for obtaining tomato ALC gene mutagenesis and replacement, in absence and presence of the homologous repair template. The average mutation frequency (72.73%) and low replacement efficiency (7.69%) were achieved in T0 transgenic plants respectively. None of homozygous mutation was detected in T0 transgenic plants, but one plant carry the heterozygous genes (Cas9/*-ALC/alc) was stably transmitted to T1 generations for segregation and genotyping. Finally, the desired alc homozygous mutants without T-DNA insertion (*/*-alc/alc) in T1 generations were acquired and further confirmed by genotype and phenotype characterization, with highlight of excellent storage performance, thus the recessive homozygous breeding elites with the character of long-shelf life were generated. Our results support that CRISPR/Cas9-induced gene replacement via HDR provides a valuable method for breeding elite innovation in tomato.

  8. A Naturally Occurring Single Amino Acid Replacement in Multiple Gene Regulator of Group A Streptococcus Significantly Increases Virulence

    PubMed Central

    Sanson, Misu; O'Neill, Brian E.; Kachroo, Priyanka; Anderson, Jeff R.; Flores, Anthony R.; Valson, Chandni; Cantu, Concepcion C.; Makthal, Nishanth; Karmonik, Christof; Fittipaldi, Nahuel; Kumaraswami, Muthiah; Musser, James M.; Olsen, Randall J.

    2016-01-01

    Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation within a species; however, few investigations demonstrate how naturally occurring SNPs may increase strain virulence. We recently used group A Streptococcus as a model pathogen to study bacteria strain genotype–patient disease phenotype relationships. Whole-genome sequencing of approximately 800 serotype M59 group A Streptococcus strains, recovered during an outbreak of severe invasive infections across North America, identified a disproportionate number of SNPs in the gene encoding multiple gene regulator of group A Streptococcus (mga). Herein, we report results of studies designed to test the hypothesis that the most commonly occurring SNP, encoding a replacement of arginine for histidine at codon 201 of Mga (H201R), significantly increases virulence. Whole transcriptome analysis revealed that the H201R replacement significantly increased expression of mga and 54 other genes, including many proven virulence factors. Compared to the wild-type strain, a H201R isogenic mutant strain caused significantly larger skin lesions in mice. Serial quantitative bacterial culture and noninvasive magnetic resonance imaging also demonstrated that the isogenic H201R strain was significantly more virulent in a nonhuman primate model of joint infection. These findings show that the H201R replacement in Mga increases the virulence of M59 group A Streptococcus and provide new insight to how a naturally occurring SNP in bacteria contributes to human disease phenotypes. PMID:25476528

  9. Cage trials using an endogenous meiotic drive gene in the mosquito Aedes aegypti to promote population replacement.

    PubMed

    Cha, Sung-Jae; Mori, Akio; Chadee, Dave D; Severson, David W

    2006-01-01

    Control of arthropod-borne diseases based on population replacement with genetically modified non-competent vectors has been proposed as a promising alternative to conventional control strategies. Due to likely fitness costs associated with vectors manipulated to carry anti-pathogen effector genes, the effector genes will need to be coupled with a strong drive system to rapidly sweep them into natural populations. Endogenous meiotic drive systems have strong and stable population replacement potential, and have previously been reported in two mosquito species: Aedes aegypti and Culex pipiens. To investigate the influence of an endogenous meiotic drive gene on Ae. aegypti population dynamics, we established three experimental population types that were initiated with 100%, 10%, and 1% male mosquitoes carrying a strong meiotic driver (T37 strain) and 100% sensitive females (RED strain), respectively. Among the 100% and 10% populations, early generations were highly male biased, which reflected the effects of the meiotic driver, and remained more than 60% male by the F(15). A genetic marker tightly linked with the meiotic driver on chromosome 1 showed strong selection for the T37 strain-specific allele. Similar but reduced effects of the meiotic driver were also observed in the 1% populations. These results suggest that release of Ae. aegypti males carrying a strong meiotic driver into drive sensitive populations can be an effective tool for population replacement, and provide a foundation for additional studies including both experimental populations and simulations by mathematical modeling.

  10. Homoeologous GSL-ELONG gene replacement for manipulation of aliphatic glucosinolates in Brassica rapa L. by marker assisted selection

    PubMed Central

    Hirani, Arvind H.; Zelmer, Carla D.; McVetty, Peter B. E.; Daayf, Fouad; Li, Genyi

    2013-01-01

    Aliphatic glucosinolates are the predominant sulfur-rich plant secondary metabolites in economically important Brassica crops. Glucosinolates and their hydrolysis products are involved in plant–microbe, plant–insect, plant–animal, and plant–human interactions. It is, therefore, important to manipulate glucosinolate profiles and contents in Brassica species. In this study, aliphatic glucosinolates were genetically manipulated through homoeologous recombination in backcross lines followed by marker assisted selection in B. rapa. A resynthesized B. napus line, from a cross between B. rapa and B. oleracea, was backcrossed with Chinese cabbage doubled haploid line, RI16. Marker assisted selection for non-functional gene was performed in each backcross generations. Advanced backcross progenies (BC3F2) were developed to identify homoeologous gene replacement and/or introgression. Reduction in 5C aliphatic glucosinolates (gluconapoleiferin, glucoalyssin, and glucobrassicanapin) was observed in BC3F2 progenies of the recurrent parent that carried the GSL-ELONG- gene. The GSL-ELONG- positive backcross progenies were also screened by the A-genome and BraGSL-ELONG gene specific marker, which linked with 5C aliphatic glucosinolates. The A-genome specific marker was absent in the plants of advanced backcross progenies which showed reduction in 5C aliphatic glucosinolates. The results suggest that the functional allele had been replaced by the non-functional GSL-ELONG- allele from B. oleracea. Some advanced backcross progenies (BC3F2) positive for the GSL-ELONG- allele and the A-genome specific SCAR marker BraMAM1-1 did not show reduction in 5C aliphatic glucosinolates, suggesting that GSL-ELONG- allele is recessive. Replacement of the functional locus in the A-genome by non-functional counterpart in the C-genome reduced the content of 5C aliphatic glucosinolates in B. rapa seeds with 20 μmol/g. PMID:23532458

  11. Novel System for Efficient Isolation of Clostridium Double-Crossover Allelic Exchange Mutants Enabling Markerless Chromosomal Gene Deletions and DNA Integration

    PubMed Central

    Al-Hinai, Mohab A.; Fast, Alan G.

    2012-01-01

    Isolation of Clostridium mutants based on gene replacement via allelic exchange remains a major limitation for this important genus. Use of a heterologous counterselection marker can facilitate the identification of the generally rare allelic exchange events. We report on the development of an inducible counterselection marker and describe its utility and broad potential in quickly and efficiently generating markerless DNA deletions and integrations at any genomic locus without the need for auxotrophic mutants or the use of the mobile group II introns. This system is based on a codon-optimized mazF toxin gene from Escherichia coli under the control of a lactose-inducible promoter from Clostridium perfringens. This system is potentially applicable to almost all members of the genus Clostridium due to their similarly low genomic GC content and comparable codon usage. We isolated all allelic-exchange-based gene deletions (ca_p0167, sigF, and sigK) or disruptions (ca_p0157 and sigF) we attempted and integrated a 3.6-kb heterologous DNA sequence (made up of a Clostridium ljungdahlii 2.1-kb formate dehydrogenase [fdh] gene plus a FLP recombination target [FRT]-flanked thiamphenicol resistance marker) into the Clostridium acetobutylicum chromosome. Furthermore, we report on the development of a plasmid system with inducible segregational instability, thus enabling efficient deployment of the FLP-FRT system to generate markerless deletion or integration mutants. This enabled expeditious deletion of the thiamphenicol resistance marker from the fdh integrant strain as well as the sigK deletion strain. More generally, our system can potentially be applied to other organisms with underdeveloped genetic tools. PMID:22983967

  12. Mytilus mitochondrial DNA contains a functional gene for a tRNASer(UCN) with a dihydrouridine arm-replacement loop and a pseudo-tRNASer(UCN) gene.

    PubMed Central

    Beagley, C T; Okimoto, R; Wolstenholme, D R

    1999-01-01

    A 2500-nucleotide pair (ntp) sequence of F-type mitochondrial (mt) DNA of the Pacific Rim mussel Mytilus californianus (class Bivalvia, phylum Mollusca) that contains two complete (ND2 and ND3) and two partial (COI and COIII) protein genes and nine tRNA genes is presented. Seven of the encoded tRNAs (Ala, Arg, His, Met(AUA), Pro, Ser(UCN), and Trp) have the potential to fold into the orthodox four-armed tRNA secondary structure, while two [tRNASer(AGN) and a second tRNASer(UCN)] will fold only into tRNAs with a dihydrouridine (DHU) arm-replacement loop. Comparison of these mt-tRNA gene sequences with previously published, corresponding M. edulis F-type mtDNA indicates that similarity between the four-armed tRNASer(UCN) genes is only 63.8% compared with an average of 92.1% (range 86.2-98. 5%) for the remaining eight tRNA genes. Northern blot analysis indicated that mature tRNAs encoded by the DHU arm-replacement loop-containing tRNASer(UCN), tRNASer(AGN), tRNAMet(AUA), tRNATrp, and tRNAPro genes occur in M. californianus mitochondria, strengthening the view that all of these genes are functional. However, Northern blot and 5' RACE (rapid amplification of cDNA ends) analyses indicated that the four-armed tRNASer(UCN) gene is transcribed into a stable RNA that includes the downstream COI sequence and is not processed into a mature tRNA. On the basis of these observations the M. californianus and M. edulis four-armed tRNASer(UCN) sequences are interpreted as pseudo-tRNASer(UCN) genes. PMID:10353906

  13. Counterselection of prokaryotic ribosomal RNA during reverse transcription using non-random hexameric oligonucleotides.

    PubMed

    Gonzalez, J M; Robb, F T

    2007-12-01

    Ribosomal RNA (rRNA) is the major component in total RNA extracts, interfering with the synthesis of cDNA corresponding to messenger RNA (mRNA). In this study, we present a novel strategy for selectively discriminating against rRNA and favoring mRNA from prokaryotes during synthesis of cDNA by reverse transcriptase. Our technique is based on the fact that rRNA sequences, in many species, are G+C rich relative to the genome at large, and highly conserved among prokaryotes. The sequence TTTT is therefore rarely found in rRNA sequences. However, TTTT priming sites are found at a much higher frequency in protein-encoding gene sequences. We designed specific hexamers (HD/DHTTTT) to prime reverse transcription reactions resulting in a selective synthesis of cDNA corresponding to mRNA from prokaryotic total RNA extractions.

  14. Large number of replacement polymorphisms in rapidly evolving genes of Drosophila. Implications for genome-wide surveys of DNA polymorphism.

    PubMed Central

    Schmid, K J; Nigro, L; Aquadro, C F; Tautz, D

    1999-01-01

    We present a survey of nucleotide polymorphism of three novel, rapidly evolving genes in populations of Drosophila melanogaster and D. simulans. Levels of silent polymorphism are comparable to other loci, but the number of replacement polymorphisms is higher than that in most other genes surveyed in D. melanogaster and D. simulans. Tests of neutrality fail to reject neutral evolution with one exception. This concerns a gene located in a region of high recombination rate in D. simulans and in a region of low recombination rate in D. melanogaster, due to an inversion. In the latter case it shows a very low number of polymorphisms, presumably due to selective sweeps in the region. Patterns of nucleotide polymorphism suggest that most substitutions are neutral or nearly neutral and that weak (positive and purifying) selection plays a significant role in the evolution of these genes. At all three loci, purifying selection of slightly deleterious replacement mutations appears to be more efficient in D. simulans than in D. melanogaster, presumably due to different effective population sizes. Our analysis suggests that current knowledge about genome-wide patterns of nucleotide polymorphism is far from complete with respect to the types and range of nucleotide substitutions and that further analysis of differences between local populations will be required to understand the forces more completely. We note that rapidly diverging and nearly neutrally evolving genes cannot be expected only in the genome of Drosophila, but are likely to occur in large numbers also in other organisms and that their function and evolution are little understood so far. PMID:10581279

  15. Enhanced gene replacements in Ku80 disruption mutants of the dermatophyte, Trichophyton mentagrophytes.

    PubMed

    Yamada, Tsuyoshi; Makimura, Koichi; Hisajima, Tatsuya; Ishihara, Yumiko; Umeda, Yoshiko; Abe, Shigeru

    2009-09-01

    The frequency of targeted gene disruption via homologous recombination is low in the clinically important dermatophyte, Trichophyton mentagrophytes. The Ku genes, Ku70 and Ku80, encode key components of the nonhomologous end-joining pathway involved in DNA double-strand break repair. Their deletion increases the homologous recombination frequency, facilitating targeted gene disruption. To improve the homologous recombination frequency in T. mentagrophytes, the Ku80 ortholog was inactivated. The nucleotide sequence of the Ku80 locus containing a 2788-bp ORF encoding a predicted product of 728 amino acids was identified, and designated as TmKu80. The predicted TmKu80 product showed a high degree of amino acid sequence similarity to known fungal Ku80 proteins. Ku80 disruption mutant strains of T. mentagrophytes were constructed by Agrobacterium tumefaciens-mediated genetic transformation. The average homologous recombination frequency was 73.3 +/- 25.2% for the areA/nit-2-like nitrogen regulatory gene (tnr) in Ku80(-) mutants, about 33-fold higher than that in wild-type controls. A high frequency (c. 67%) was also obtained for the Tri m4 gene encoding a putative serine protease. Ku80(-) mutant strains will be useful for large-scale reverse genetics studies of dermatophytes, including T. mentagrophytes, providing valuable information on the basic mechanisms of host invasion.

  16. Insect glycerol transporters evolved by functional co-option and gene replacement.

    PubMed

    Finn, Roderick Nigel; Chauvigné, François; Stavang, Jon Anders; Belles, Xavier; Cerdà, Joan

    2015-07-17

    Transmembrane glycerol transport is typically facilitated by aquaglyceroporins in Prokaryota and Eukaryota. In holometabolan insects however, aquaglyceroporins are absent, yet several species possess polyol permeable aquaporins. It thus remains unknown how glycerol transport evolved in the Holometabola. By combining phylogenetic and functional studies, here we show that a more efficient form of glycerol transporter related to the water-selective channel AQP4 specifically evolved and multiplied in the insect lineage, resulting in the replacement of the ancestral branch of aquaglyceroporins in holometabolan insects. To recapitulate this evolutionary process, we generate specific mutants in distantly related insect aquaporins and human AQP4 and show that a single mutation in the selectivity filter converted a water-selective channel into a glycerol transporter at the root of the crown clade of hexapod insects. Integration of phanerozoic climate models suggests that these events were associated with the emergence of complete metamorphosis and the unparalleled radiation of insects.

  17. Insect glycerol transporters evolved by functional co-option and gene replacement

    PubMed Central

    Finn, Roderick Nigel; Chauvigné, François; Stavang, Jon Anders; Belles, Xavier; Cerdà, Joan

    2015-01-01

    Transmembrane glycerol transport is typically facilitated by aquaglyceroporins in Prokaryota and Eukaryota. In holometabolan insects however, aquaglyceroporins are absent, yet several species possess polyol permeable aquaporins. It thus remains unknown how glycerol transport evolved in the Holometabola. By combining phylogenetic and functional studies, here we show that a more efficient form of glycerol transporter related to the water-selective channel AQP4 specifically evolved and multiplied in the insect lineage, resulting in the replacement of the ancestral branch of aquaglyceroporins in holometabolan insects. To recapitulate this evolutionary process, we generate specific mutants in distantly related insect aquaporins and human AQP4 and show that a single mutation in the selectivity filter converted a water-selective channel into a glycerol transporter at the root of the crown clade of hexapod insects. Integration of phanerozoic climate models suggests that these events were associated with the emergence of complete metamorphosis and the unparalleled radiation of insects. PMID:26183829

  18. Gene therapy/bone marrow transplantation in ADA-deficient mice: roles of enzyme-replacement therapy and cytoreduction.

    PubMed

    Carbonaro, Denise A; Jin, Xiangyang; Wang, Xingchao; Yu, Xiao-Jin; Rozengurt, Nora; Kaufman, Michael L; Wang, Xiaoyan; Gjertson, David; Zhou, Yang; Blackburn, Michael R; Kohn, Donald B

    2012-11-01

    Gene therapy (GT) for adenosine deaminase-deficient severe combined immune deficiency (ADA-SCID) can provide significant long-term benefit when patients are given nonmyeloablative conditioning and ADA enzyme-replacement therapy (ERT) is withheld before autologous transplantation of γ-retroviral vector-transduced BM CD34+ cells. To determine the contributions of conditioning and discontinuation of ERT to the therapeutic effects, we analyzed these factors in Ada gene knockout mice (Ada(-/-)). Mice were transplanted with ADA-deficient marrow transduced with an ADA-expressing γ-retroviral vector without preconditioning or after 200 cGy or 900 cGy total-body irradiation and evaluated after 4 months. In all tissues analyzed, vector copy numbers (VCNs) were 100- to 1000-fold greater in mice receiving 900 cGy compared with 200 cGy (P < .05). In mice receiving 200 cGy, VCN was similar whether ERT was stopped or given for 1 or 4 months after GT. In unconditioned mice, there was decreased survival with and without ERT, and VCN was very low to undetectable. When recipients were conditioned with 200 cGy and received transduced lineage-depleted marrow, only recipients receiving ERT (1 or 4 months) had detectable vector sequences in thymocytes. In conclusion, cytoreduction is important for the engraftment of gene-transduced HSC, and short-term ERT after GT did not diminish the capacity of gene-corrected cells to engraft and persist.

  19. Discovery of the rpl10 Gene in Diverse Plant Mitochondrial Genomes and Its Probable Replacement by the Nuclear Gene for Chloroplast RPL10 in Two Lineages of Angiosperms

    PubMed Central

    Kubo, Nakao; Arimura, Shin-ichi

    2010-01-01

    Mitochondrial genomes of plants are much larger than those of mammals and often contain conserved open reading frames (ORFs) of unknown function. Here, we show that one of these conserved ORFs is actually the gene for ribosomal protein L10 (rpl10) in plant. No rpl10 gene has heretofore been reported in any mitochondrial genome other than the exceptionally gene-rich genome of the protist Reclinomonas americana. Conserved ORFs corresponding to rpl10 are present in a wide diversity of land plant and green algal mitochondrial genomes. The mitochondrial rpl10 genes are transcribed in all nine land plants examined, with five seed plant genes subject to RNA editing. In addition, mitochondrial-rpl10-like cDNAs were identified in EST libraries from numerous land plants. In three lineages of angiosperms, rpl10 is either lost from the mitochondrial genome or a pseudogene. In two of them (Brassicaceae and monocots), no nuclear copy of mitochondrial rpl10 is identifiably present, and instead a second copy of nuclear-encoded chloroplast rpl10 is present. Transient assays using green fluorescent protein indicate that this duplicate gene is dual targeted to mitochondria and chloroplasts. We infer that mitochondrial rpl10 has been functionally replaced by duplicated chloroplast counterparts in Brassicaceae and monocots. PMID:19934175

  20. A copper-responsive promoter replacement system to investigate gene functions in Trichoderma reesei: a case study in characterizing SAGA genes.

    PubMed

    Zheng, Fanglin; Cao, Yanli; Lv, Xinxing; Wang, Lei; Li, Chunyan; Zhang, Weixin; Chen, Guanjun; Liu, Weifeng

    2017-03-01

    Trichoderma reesei represents an important workhorse for industrial production of cellulases as well as other proteins. The molecular mechanism underlying the regulation of cellulase production as well as other physiological processes in T. reesei is still insufficiently understood. We constructed a P tcu1 -based promoter substitution cassette that allowed one-step replacement of the endogenous promoter for controlling the target gene expression with copper. We then showed that copper repression of the histone acetyltransferase gene gcn5 phenocopied the gcn5 deletion strain. Using the same strategy, we further characterized the function of another putative Spt-Ada-Gcn5 acetyltransferase (SAGA) complex subunit encoding gene, ada2, in T. reesei. Similar to the repression of gcn5, the addition of copper to the P tcu1 -ada2 strain not only drastically reduced the vegetative growth and conidiation in T. reesei but also severely compromised the induced cellulase gene expression. The developed strategy will thus be potentially useful to probe the biological function of the large fraction of T. reesei genes with unknown functions including those essential genes in the genome to expand its extraordinary biotechnological potential.

  1. Discovery of the rpl10 gene in diverse plant mitochondrial genomes and its probable replacement by the nuclear gene for chloroplast RPL10 in two lineages of angiosperms.

    PubMed

    Kubo, Nakao; Arimura, Shin-ichi

    2010-02-01

    Mitochondrial genomes of plants are much larger than those of mammals and often contain conserved open reading frames (ORFs) of unknown function. Here, we show that one of these conserved ORFs is actually the gene for ribosomal protein L10 (rpl10) in plant. No rpl10 gene has heretofore been reported in any mitochondrial genome other than the exceptionally gene-rich genome of the protist Reclinomonas americana. Conserved ORFs corresponding to rpl10 are present in a wide diversity of land plant and green algal mitochondrial genomes. The mitochondrial rpl10 genes are transcribed in all nine land plants examined, with five seed plant genes subject to RNA editing. In addition, mitochondrial-rpl10-like cDNAs were identified in EST libraries from numerous land plants. In three lineages of angiosperms, rpl10 is either lost from the mitochondrial genome or a pseudogene. In two of them (Brassicaceae and monocots), no nuclear copy of mitochondrial rpl10 is identifiably present, and instead a second copy of nuclear-encoded chloroplast rpl10 is present. Transient assays using green fluorescent protein indicate that this duplicate gene is dual targeted to mitochondria and chloroplasts. We infer that mitochondrial rpl10 has been functionally replaced by duplicated chloroplast counterparts in Brassicaceae and monocots.

  2. Replication inhibition by nucleoside analogues of a recombinant Autographa californica multicapsid nuclear polyhedrosis virus harboring the herpes thymidine kinase gene driven by the IE-1(0) promoter: a new way to select recombinant baculoviruses.

    PubMed Central

    Godeau, F; Saucier, C; Kourilsky, P

    1992-01-01

    The expression of the thymidine-thymidylate kinase (HSV1-TK), (ATP: thymidine 5'-phosphotransferase; EC 2.7.1.21) of herpes simplex virus type 1 endows the host cell with a conditional lethal phenotype which depends on the presence of nucleoside analogues metabolized by this enzyme into toxic inhibitors of DNA replication. To generate a recombinant baculovirus that could be selected against by nucleoside analogs, the HSV1-tk coding sequence was placed under the control of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) immediate early promoterm IE-1(0), and this construction was introduced via homologous recombination into the polyhedrin locus of AcMNPV. Two recombinant baculoviruses harboring this gene construct at the polyhedrin locus were isolated and tested for their ability to replicate in the presence of various concentrations of the nucleoside analog 9-(1,3-Dihydroxy-2-propoxymethyl)guanine (Ganciclovir). Neither Sf9 lepidopteran cell viability nor replication of wild type or beta-Galactosidase-expressing recombinant AcMNPVs were affected by concentrations of Ganciclovir up to 100 microM. In contrast, replication of the recombinant AcMNPV virus harboring the HSV1-tk gene was inhibited by Ganciclovir in a dose-dependent manner. The inhibition was detectable at 2 microM and complete at 100 microM. This property was exploited in model isolations aimed at purifying new recombinant viruses having lost this counter-selectable gene marker as a result of homologous recombination at the polyhedrin locus after cotransfection of the viral DNA with a replacement vector. After being propagated in the presence of Ganciclovir, the progeny of such co-transfections contained over 85% recombinant viruses, demonstrating that counter-selection of parental HSV1-tk-containing viruses by Ganciclovir constitutes a novel approach for recombinant baculovirus isolation. Images PMID:1335569

  3. Invasions by Eurasian Avian Influenza Virus H6 Genes and Replacement of Its North American Clade

    PubMed Central

    Li, Jinling; Cardona, Carol J.; Miller, Joy; Carpenter, Tim E.

    2009-01-01

    The spread of highly pathogenic avian influenza virus (AIV) (H5N1) underlines the potential for global AIV movement through birds. The phylogenies of AIV genes from avian hosts usually separate into Eurasian and North American clades, reflecting limited bird migration between the hemispheres. However, mounting evidence that some H6 sequences from North America cluster with Eurasian subtype H6 sequences calls the strict hemispheric divide into question. We conducted a comprehensive phylogenetic analysis of the extent and timing of cross-hemisphere movements by the H6 gene. Results suggested that Eurasian H6 subtype has invaded North America several times, with the first invasions occurring 10 years before the first detection of invading isolates. The members of the North American clade decreased from 100% in the 1980s to 20% in the 2000s among H6 isolates from North America. Unraveling the reasons for this large-scale gene movement between hemispheres might identify drivers of global AIV circulation. PMID:19624918

  4. Gene replacement reveals that p115/SNARE interactions are essential for Golgi biogenesis

    PubMed Central

    Puthenveedu, Manojkumar A.; Linstedt, Adam D.

    2004-01-01

    Functional characterization of protein interactions in mammalian systems has been hindered by the inability to perform complementation analyses in vivo. Here, we use functional replacement of the vesicle docking protein p115 to separate its essential from its nonessential interactions. p115 is required for biogenesis of the Golgi apparatus, but it is unclear whether its mechanism of action requires its golgin and/or SNARE interactions. Short interfering RNA-mediated knockdown of p115 induced extensive Golgi fragmentation and impaired secretory traffic. Reassembly of a structurally and functionally normal Golgi occurred on expression of a p115 homologue not recognized by the short interfering RNA. Strikingly, versions of p115 lacking its phosphorylation site and the golgin-binding domains also restored the Golgi apparatus in cells lacking endogenous p115. In contrast, the p115 SNARE-interacting domain was required for Golgi biogenesis. This suggests that p115 acts directly, rather than via a tether, to catalyze trans-SNARE complex formation preceding membrane fusion. PMID:14736916

  5. Alleles of the maize P gene with distinct tissue specificities encode Myb-homologous proteins with C-terminal replacements.

    PubMed Central

    Chopra, S; Athma, P; Peterson, T

    1996-01-01

    The maize P gene is a transcriptional regulator of genes encoding enzymes for flavonoid biosynthesis in the pathway leading to the production of a red phlobaphene pigment. Multiple alleles of the P gene confer distinct patterns of pigmentation to specific floral organs, such as the kernel pericarp and cob tissues. To determine the basis of allele-specific pigmentation, we have characterized the gene products and transcript accumulation patterns of the P-wr allele, which specifies colorless pericarps and red cob tissues. RNA transcripts of P-wr are present in colorless pericarps as well as in the colored cob tissues; however, the expression of P-wr in pericarp does not induce the accumulation of transcripts from the C2 and A1 genes, which encode enzymes for flavonoid pigment biosynthesis. The coding sequences of P-wr were compared with the P-rr allele, which specifies red pericarp and red cob. The P-wr and P-rr cDNA sequences are very similar in their 5' regions. There are only two nucleotide changes that result in amino acid differences; both are outside of the Myb-homologous DNA binding domain. In contrast, the 3' coding region of P-rr is replaced by a unique 210-bp sequence in P-wr. The predicted P-wr protein has a C-terminal sequence resembling a cysteine-containing metal binding domain that is not present in the P-rr protein. These results indicate that the differential pericarp pigmentation specified by the P-rr and P-wr alleles does not result from an absence of P-wr transcripts in pericarps. Rather, the allele-specific patterns of P-rr and P-wr pigmentation may be associated with structural differences in the proteins encoded by each allele. PMID:8768374

  6. BRaf signaling principles unveiled by large-scale human mutation analysis with a rapid lentivirus-based gene replacement method.

    PubMed

    Lim, Chae-Seok; Kang, Xi; Mirabella, Vincent; Zhang, Huaye; Bu, Qian; Araki, Yoichi; Hoang, Elizabeth T; Wang, Shiqiang; Shen, Ying; Choi, Sukwoo; Kaang, Bong-Kiun; Chang, Qiang; Pang, Zhiping P; Huganir, Richard L; Zhu, J Julius

    2017-03-15

    Rapid advances in genetics are linking mutations on genes to diseases at an exponential rate, yet characterizing the gene mutation-cell behavior relationships essential for precision medicine remains a daunting task. More than 350 mutations on small GTPase BRaf are associated with various tumors, and ∼40 mutations are associated with the neurodevelopmental disorder cardio-facio-cutaneous syndrome (CFC). We developed a fast cost-effective lentivirus-based rapid gene replacement method to interrogate the physiopathology of BRaf and ∼50 disease-linked BRaf mutants, including all CFC-linked mutants. Analysis of simultaneous multiple patch-clamp recordings from 6068 pairs of rat neurons with validation in additional mouse and human neurons and multiple learning tests from 1486 rats identified BRaf as the key missing signaling effector in the common synaptic NMDA-R-CaMKII-SynGap-Ras-BRaf-MEK-ERK transduction cascade. Moreover, the analysis creates the original big data unveiling three general features of BRaf signaling. This study establishes the first efficient procedure that permits large-scale functional analysis of human disease-linked mutations essential for precision medicine. © 2017 Lim et al.; Published by Cold Spring Harbor Laboratory Press.

  7. Potential of Advexin: a p53 gene-replacement therapy in Li-Fraumeni syndrome.

    PubMed

    Nemunaitis, Jackie M; Nemunaitis, John

    2008-12-01

    Li-Fraumeni syndrome is a rare autosomal dominant cancer predisposition syndrome. The majority of families fulfilling definition of Li-Fraumeni syndrome demonstrate inherited abnormalities involving the p53 gene. Cells with dysfunctional p53 are predisposed to the development of cancer phenotype. Advexin (Introgen Therapeutics Inc., TX, USA) is an adenoviral-based experimental therapeutic that provides delivery of wild-type p53 to cancer cells and demonstrates anticancer activity following adequate expression of p53. Theoretically, correction of p53 function in cancer developing in patients with Li-Fraumeni syndrome through treatment with Advexin will provide anti-tumor activity. One patient with Li-Fraumeni syndrome has been reported to have responded to Advexin. This review will summarize background knowledge of Li-Fraumeni syndrome, mechanisms of Advexin and clinical response of cancer to Advexin with a focus on Li-Fraumeni syndrome.

  8. Long-term rescue of cone photoreceptor degeneration in retinitis pigmentosa 2 (RP2)-knockout mice by gene replacement therapy.

    PubMed

    Mookherjee, Suddhasil; Hiriyanna, Suja; Kaneshiro, Kayleigh; Li, Linjing; Li, Yichao; Li, Wei; Qian, Haohua; Li, Tiansen; Khanna, Hemant; Colosi, Peter; Swaroop, Anand; Wu, Zhijian

    2015-11-15

    Retinal neurodegenerative diseases are especially attractive targets for gene replacement therapy, which appears to be clinically effective for several monogenic diseases. X-linked forms of retinitis pigmentosa (XLRP) are relatively severe blinding disorders, resulting from progressive photoreceptor dysfunction primarily caused by mutations in RPGR or RP2 gene. With a goal to develop gene therapy for the XLRP-RP2 disease, we first performed detailed characterization of the Rp2-knockout (Rp2-KO) mice and observed early-onset cone dysfunction, which was followed by progressive cone degeneration, mimicking cone vision impairment in XLRP patients. The mice also exhibited distinct and significantly delayed falling phase of photopic b-wave of electroretinogram (ERG). Concurrently, we generated a self-complementary adeno-associated viral (AAV) vector carrying human RP2-coding sequence and demonstrated its ability to mediate stable RP2 protein expression in mouse photoreceptors. A long-term efficacy study was then conducted in Rp2-KO mice following AAV-RP2 vector administration. Preservation of cone function was achieved with a wide dose range over 18-month duration, as evidenced by photopic ERG and optomotor tests. The slower b-wave kinetics was also completely restored. Morphologically, the treatment preserved cone viability, corrected mis-trafficking of M-cone opsin and restored cone PDE6 expression. The therapeutic effect was achieved even in mice that received treatment at an advanced disease stage. The highest AAV-RP2 dose group demonstrated retinal toxicity, highlighting the importance of careful vector dosing in designing future human trials. The wide range of effective dose, a broad treatment window and long-lasting therapeutic effects should make the RP2 gene therapy attractive for clinical development. Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  9. Genetic rescue of segmentation defect in MesP2-deficient mice by MesP1 gene replacement.

    PubMed

    Saga, Y

    1998-07-01

    Gene knock-out and knock-in strategies are employed to investigate the function of MesP1. MesP1 belongs to the same family of bHLH transcription factors as MesP2. The early expression pattern observed in the early mesoderm at the onset of gastrulation is restricted to Mesp1, while the later expression pattern in the anterior presomitic mesoderm during somitogenesis is almost the same for Mesp1 as for Mesp2. Homozygous Mesp1 null mice exhibited growth retardation after 7.5 dpc and died before 10.5 dpc with many developmental defects. The function of MesP1 during somitogenesis was not clearly revealed because of their early death and the possible compensation by MesP2. In order to examine the functions of MesP1 during somitogenesis, we replaced the Mesp2 gene with Mesp1 cDNA, using a gene knock-in strategy. The introduced Mesp1 cDNA could rescue the defects caused by Mesp2 deficiency in a dosage-dependent manner. Mice which lacked Mesp2 expression but had four copies of the Mesp1 gene survived into the adulthood and were fertile. The skeletal defects and the reduction in expression of Notch1, Notch2 and FGFR-1 previously observed in Mesp2 null mice were almost completely rescued by the introduced MesP1. Thus, it is concluded that the functions of MesP1 during somitogenesis, like MesP2, are also mediated via notch-delta and FGF signaling systems.

  10. Long-term rescue of cone photoreceptor degeneration in retinitis pigmentosa 2 (RP2)-knockout mice by gene replacement therapy

    PubMed Central

    Mookherjee, Suddhasil; Hiriyanna, Suja; Kaneshiro, Kayleigh; Li, Linjing; Li, Yichao; Li, Wei; Qian, Haohua; Li, Tiansen; Khanna, Hemant; Colosi, Peter; Swaroop, Anand; Wu, Zhijian

    2015-01-01

    Retinal neurodegenerative diseases are especially attractive targets for gene replacement therapy, which appears to be clinically effective for several monogenic diseases. X-linked forms of retinitis pigmentosa (XLRP) are relatively severe blinding disorders, resulting from progressive photoreceptor dysfunction primarily caused by mutations in RPGR or RP2 gene. With a goal to develop gene therapy for the XLRP-RP2 disease, we first performed detailed characterization of the Rp2-knockout (Rp2-KO) mice and observed early-onset cone dysfunction, which was followed by progressive cone degeneration, mimicking cone vision impairment in XLRP patients. The mice also exhibited distinct and significantly delayed falling phase of photopic b-wave of electroretinogram (ERG). Concurrently, we generated a self-complementary adeno-associated viral (AAV) vector carrying human RP2-coding sequence and demonstrated its ability to mediate stable RP2 protein expression in mouse photoreceptors. A long-term efficacy study was then conducted in Rp2-KO mice following AAV-RP2 vector administration. Preservation of cone function was achieved with a wide dose range over 18-month duration, as evidenced by photopic ERG and optomotor tests. The slower b-wave kinetics was also completely restored. Morphologically, the treatment preserved cone viability, corrected mis-trafficking of M-cone opsin and restored cone PDE6 expression. The therapeutic effect was achieved even in mice that received treatment at an advanced disease stage. The highest AAV-RP2 dose group demonstrated retinal toxicity, highlighting the importance of careful vector dosing in designing future human trials. The wide range of effective dose, a broad treatment window and long-lasting therapeutic effects should make the RP2 gene therapy attractive for clinical development. PMID:26358772

  11. Structural recovery of the retina in a retinoschisin-deficient mouse after gene replacement therapy by solid lipid nanoparticles.

    PubMed

    Apaolaza, P S; Del Pozo-Rodríguez, A; Solinís, M A; Rodríguez, J M; Friedrich, U; Torrecilla, J; Weber, B H F; Rodríguez-Gascón, A

    2016-06-01

    X-linked juvenile retinoschisis (XLRS) is a retinal degenerative disorder caused by mutations in the RS1 gene encoding a protein termed retinoschisin. The disease is an excellent candidate for gene replacement therapy as the majority of mutations have been shown to lead to a complete deficiency of the secreted protein in the retinal structures. In this work, we have studied the ability of non-viral vectors based on solid lipid nanoparticles (SLN) to induce the expression of retinoschisin in photoreceptors (PR) after intravitreal administration to Rs1h-deficient mice. We designed two vectors prepared with SLN, protamine, and dextran (DX) or hyaluronic acid (HA), bearing a plasmid containing the human RS1 gene under the control of the murin opsin promoter (mOPS). In vitro, the nanocarriers were able to induce the expression of retinoschisin in a PR cell line. After injection into the murine vitreous, the formulation prepared with HA induced a higher transfection level in PR than the formulation prepared with DX. Moreover, the level of retinoschisin in the inner nuclear layer (INL), where bipolar cells are located, was also higher. Two weeks after vitreal administration into Rs1h-deficient mice, both formulations showed significant improvement of the retinal structure by inducing a decrease of cavities and PR loss, and an increase of retinal and outer nuclear layer (ONL) thickness. HA-SLN resulted in a significant higher increase in the thickness of both retina and ONL, which can be explained by the higher transfection level of PR. In conclusion, we have shown the structural improvement of the retina of Rs1h-deficient mice with PR specific expression of the RS1 gene driven by the specific promoter mOPS, after successful delivery via SLN-based non-viral vectors.

  12. Three-parent in vitro fertilization: gene replacement for the prevention of inherited mitochondrial diseases.

    PubMed

    Amato, Paula; Tachibana, Masahito; Sparman, Michelle; Mitalipov, Shoukhrat

    2014-01-01

    The exchange of nuclear genetic material between oocytes and embryos offers a novel reproductive option for the prevention of inherited mitochondrial diseases. Mitochondrial dysfunction has been recognized as a significant cause of a number of serious multiorgan diseases. Tissues with a high metabolic demand, such as brain, heart, muscle, and central nervous system, are often affected. Mitochondrial disease can be due to mutations in mitochondrial DNA or in nuclear genes involved in mitochondrial function. There is no curative treatment for patients with mitochondrial disease. Given the lack of treatments and the limitations of prenatal and preimplantation diagnosis, attention has focused on prevention of transmission of mitochondrial disease through germline gene replacement therapy. Because mitochondrial DNA is strictly maternally inherited, two approaches have been proposed. In the first, the nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote. A second technique involves transfer of the metaphase II spindle from the unfertilized oocyte of an affected woman to an enucleated donor oocyte. Our group recently reported successful spindle transfer between human oocytes, resulting in blastocyst development and embryonic stem cell derivation, with very low levels of heteroplasmy. In this review we summarize these novel assisted reproductive techniques and their use to prevent transmission of mitochondrial disorders. The promises and challenges are discussed, focusing on their potential clinical application.

  13. S-opsin knockout mice with the endogenous M opsin gene replaced by an L opsin variant

    PubMed Central

    Greenwald, Scott H.; Kuchenbecker, James A.; Roberson, Daniel K.; Neitz, Maureen; Neitz, Jay

    2014-01-01

    Specific variants of human long-wavelength (L) and middle-wavelength (M) cone opsin genes have recently been associated with a variety of vision disorders caused by cone malfunction, including red-green color vision deficiency, blue cone monochromacy, myopia, and cone dystrophy. Strikingly, unlike disease-causing mutations in rhodopsin, most of the cone opsin alleles that are associated with vision disorders do not have deleterious point mutations. Instead, specific combinations of normal polymorphisms that arose by genetic recombination between the genes encoding L and M opsins appear to cause disease. Knockout/knock-in mice promise to make it possible to study how these deleterious cone opsin variants affect the structure, function, and viability of the cone photoreceptors. Ideally, we would like to evaluate different variants that cause vision disorders in humans against a control pigment that is not associated with vision disorders, and each variant should be expressed as the sole photopigment in each mouse cone, as is the case in humans. To evaluate the feasibility of this approach, we created a line of mice to serve as the control in the analysis of disease causing mutations by replacing exon 2 through 6 of the mouse M opsin gene with the corresponding cDNA for a human L opsin variant that is associated with normal vision. Experiments reported here establish that the resulting pigment, which differs from the endogenous mouse M opsin at 35 amino acid positions, functions normally in mouse cones. This pigment was evaluated in mice with and without co-expression of the mouse short wavelength (S) opsin. Here, the creation and validation of two lines of genetically engineered mice that can be used to study disease-causing variants of human L/M-opsins, in vivo, are described. PMID:24801621

  14. S-opsin knockout mice with the endogenous M-opsin gene replaced by an L-opsin variant.

    PubMed

    Greenwald, Scott H; Kuchenbecker, James A; Roberson, Daniel K; Neitz, Maureen; Neitz, Jay

    2014-01-01

    Specific variants of human long-wavelength (L) and middle-wavelength (M) cone opsin genes have recently been associated with a variety of vision disorders caused by cone malfunction, including red-green color vision deficiency, blue cone monochromacy, myopia, and cone dystrophy. Strikingly, unlike disease-causing mutations in rhodopsin, most of the cone opsin alleles that are associated with vision disorders do not have deleterious point mutations. Instead, specific combinations of normal polymorphisms that arose by genetic recombination between the genes encoding L and M opsins appear to cause disease. Knockout/knock-in mice promise to make it possible to study how these deleterious cone opsin variants affect the structure, function, and viability of the cone photoreceptors. Ideally, we would like to evaluate different variants that cause vision disorders in humans against a control pigment that is not associated with vision disorders, and each variant should be expressed as the sole photopigment in each mouse cone, as is the case in humans. To evaluate the feasibility of this approach, we created a line of mice to serve as the control in the analysis of disease-causing mutations by replacing exon 2 through 6 of the mouse M-opsin gene with the corresponding cDNA for a human L-opsin variant that is associated with normal vision. Experiments reported here establish that the resulting pigment, which differs from the endogenous mouse M opsin at 35 amino acid positions, functions normally in mouse cones. This pigment was evaluated in mice with and without coexpression of the mouse short wavelength (S) opsin. Here, the creation and validation of two lines of genetically engineered mice that can be used to study disease-causing variants of human L/M-opsins, in vivo, are described.

  15. Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis

    PubMed Central

    Castro-Bravo, Nuria; Hidalgo-Cantabrana, Claudio; Rodriguez-Carvajal, Miguel A.; Ruas-Madiedo, Patricia; Margolles, Abelardo

    2017-01-01

    An extracellular layer of exopolysaccharides (EPS) covers the surface of some Bifidobacterium animalis subsp. lactis strains, which could be of relevance for its probiotic performance. In order to understand the functional characteristics of B. animalis subsp. lactis, two isogenic strains that differ in their EPS-producing phenotype, due to a single mutation in the gene Balat_1410, were studied. By means of a double crossover recombination strategy, successfully used for the first time in bifidobacteria, Balat_1410 in the type strain B. animalis subsp. lactis DSM10140 was replaced by a mutated gene containing a non-synonymous mutation previously associated with the appearance of a mucoid-ropy phenotype. Nuclear magnetic resonance and SEC-MALS analyses showed that the novel strain harboring the mutation acquired a ropy phenotype, due to the production of a high molecular weight (HMW)-EPS that is not produced in the wild-type strain. Fluorescence labeling of both strains with two fluorescent proteins, m-Cherry and Green Fluorescent Protein, was achieved by expressing the corresponding genes under the control of a native selected promoter (the elongation factor Tu promoter). Remarkably, qualitative and quantitative fluorescence analyses demonstrated that the ropy strain displays a lower capability to adhere to human intestinal epithelial cells. In addition, the presence of the HMW-EPS reduced the capability of the producing strain to form biofilms upon three different abiotic surfaces. This work also highlights the fact that different EPS confer variable functional characteristics to the bifidobacterial surface, which may be relevant for the performance of B. animalis subsp. lactis as a probiotic. The construction of molecular tools allowing the functional characterization of surface structures in next generation probiotics is still a challenging issue that deserves further attention, given the relevant role that such molecules must play in the interaction with the

  16. AAV-Mediated Lysophosphatidylcholine Acyltransferase 1 (Lpcat1) Gene Replacement Therapy Rescues Retinal Degeneration in rd11 Mice

    PubMed Central

    Dai, Xufeng; Han, Juanjuan; Qi, Yan; Zhang, Hua; Xiang, Lue; Lv, Jineng; Li, Jie; Deng, Wen-Tao; Chang, Bo; Hauswirth, William W.; Pang, Ji-jing

    2014-01-01

    Purpose. The retinal degeneration 11 (rd11) mouse is a newly discovered, naturally occurring animal model with early photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration. The rd11 mice carry a spontaneous mutation in the lysophosphatidylcholine acyltransferase 1 (Lpcat1) gene. Here, we evaluate whether gene replacement therapy using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can arrest retinal degeneration and restore retinal function in this model. Methods. The AAV8 (Y733F)-smCBA-Lpcat1 was delivered subretinally to postnatal day 14 (P14) rd11 mice in one eye only. At 10 weeks after injection, treated rd11 mice were examined by visually-guided behavior, electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT), and then killed for morphologic and biochemical examination. Results. Substantial scotopic and photopic ERG signals were maintained in treated rd11 eyes, whereas untreated eyes in the same animals showed extinguished signals. The SD-OCT (in vivo) and light microscopy (in vitro) showed a substantial preservation of the outer nuclear layer in most parts of the treated retina only. Almost wild-type LPCAT1 expression in photoreceptors with strong rod rhodopsin and M/S cone opsin staining, and normal visually-guided water maze behavioral performances were observed in treated rd11 mice. Conclusions. The results demonstrate that the tyrosine-capsid mutant AAV8 (Y733F) vector is effective for treating rapidly degenerating models of retinal degeneration and, moreover, is more therapeutically effective than AAV2 (Y444, 500, 730F) vector with the same promoter-cDNA payload. To our knowledge, this is the first demonstration of phenotypic rescue by gene therapy in an animal model of retinal degeneration caused by Lpcat1 mutation. PMID:24557352

  17. Improving the Efficiency of Homologous Gene Replacement by Disrupting the NHEJ Pathway for Gene KusA in the Oleaginous Fungus Mortierella alpina

    NASA Astrophysics Data System (ADS)

    Krueger, Kathleen; Dai, Ziyu; Uzuner, Uger

    2012-11-01

    Mortierella alpina, a oleaginous filamentous fungus, is one of industrial fungal strains known for the production of arachidonic acid. It is also of particular interest for hydrocarbon biofuel production since it is able to produce up to 50% of its mass in rich, long-chain polyunsaturated fatty acids [PUFA's]. In addition to high fatty acid production, M. alpina like many other oleaginous fungi, already have mechanisms for accumulating significant concentrations of hydrophobic compounds making it a naturally equipped candidate to handle potential toxic concentrations of hydrocarbons. The goal of this study was to develop an efficient transformation method for this strain, hence allowing researchers to further manipulate these fungi for further improvement of lipid production. Included was optimization of best culture medium for growth and maintenance, optimal conditions for protoplast generation, and replacement of the homologous KusA gene. A successful deletion of KusA gene within biotechnologically important M. alpina could enable homologous recombination of other genes of interest in a higher frequency. This capacity may also improve the advancing the production of microbial oils for bioenergy and arachidonic acid human health applications.

  18. The mechanism of mammalian gene replacement is consistent with the formation of long regions of heteroduplex DNA associated with two crossing-over events.

    PubMed

    Li, J; Read, L R; Baker, M D

    2001-01-01

    In this study, the mechanism of mammalian gene replacement was investigated. The system is based on detecting homologous recombination between transferred vector DNA and the haploid, chromosomal immunoglobulin mu-delta region in a murine hybridoma cell line. The backbone of the gene replacement vector (pCmuCdeltapal) consists of pSV2neo sequences bounded on one side by homology to the mu gene constant (Cmu) region and on the other side by homology to the delta gene constant (Cdelta) region. The Cmu and Cdelta flanking arms of homology were marked by insertions of an identical 30-bp palindrome which frequently escapes mismatch repair when in heteroduplex DNA (hDNA). As a result, intermediates bearing unrepaired hDNA generate mixed (sectored) recombinants following DNA replication and cell division. To monitor the presence and position of sectored sites and, hence, hDNA formation during the recombination process, the palindrome contained a unique NotI site that replaced an endogenous restriction enzyme site at each marker position in the vector-borne Cmu and Cdelta regions. Gene replacement was studied under conditions which permitted the efficient recovery of the product(s) of individual recombination events. Analysis of marker segregation patterns in independent recombinants revealed that extensive hDNA was formed within the Cmu and Cdelta regions. In several recombinants, palindrome markers in the Cmu and Cdelta regions resided on opposite DNA strands (trans configuration). These results are consistent with the mammalian gene replacement reaction involving two crossing-over events in homologous flanking DNA.

  19. Low-dose Gene Therapy Reduces the Frequency of Enzyme Replacement Therapy in a Mouse Model of Lysosomal Storage Disease

    PubMed Central

    Alliegro, Marialuisa; Ferla, Rita; Nusco, Edoardo; De Leonibus, Chiara; Settembre, Carmine; Auricchio, Alberto

    2016-01-01

    Enzyme replacement therapy (ERT) is the standard of care for several lysosomal storage diseases (LSDs). ERT, however, requires multiple and costly administrations and has limited efficacy. We recently showed that a single high dose administration of adeno-associated viral vector serotype 8 (AAV2/8) is at least as effective as weekly ERT in a mouse model of mucopolysaccharidosis type VI (MPS VI). However, systemic administration of high doses of AAV might result in both cell-mediated immune responses and insertional mutagenesis. Here we evaluated whether the combination of low doses of AAV2/8 with a less frequent (monthly) than canonical (weekly) ERT schedule may be as effective as the single treatments at high doses or frequent regimen. A greater reduction of both urinary glycosaminoglycans, considered a sensitive biomarker of therapeutic efficacy, and storage in the myocardium and heart valves was observed in mice receiving the combined than the single therapies. Importantly, these levels of correction were similar to those we obtained in a previous study following either high doses of AAV2/8 or weekly ERT. Our data show that low-dose gene therapy can be used as a means to rarify ERT administration, thus reducing both the risks and costs associated with either therapies. PMID:27658524

  20. Characterization of a Herpes Simplex Virus 1 (HSV-1) Chimera in Which the Us3 Protein Kinase Gene Is Replaced with the HSV-2 Us3 Gene.

    PubMed

    Shindo, Keiko; Kato, Akihisa; Koyanagi, Naoto; Sagara, Hiroshi; Arii, Jun; Kawaguchi, Yasushi

    2015-10-21

    Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) play important roles in viral replication and pathogenicity. To investigate type-specific differences between HSV-1 Us3 and HSV-2 Us3 in cells infected by viruses with all the same viral gene products except for their Us3 kinases, we constructed and characterized a recombinant HSV-1 in which its Us3 gene was replaced with the HSV-2 Us3 gene. Replacement of HSV-1 Us3 with HSV-2 Us3 had no apparent effect on viral growth in cell cultures or on the range of proteins phosphorylated by Us3. HSV-2 Us3 efficiently compensated for HSV-1 Us3 functions, including blocking apoptosis, controlling infected cell morphology, and downregulating cell surface expression of viral envelope glycoprotein B. In contrast, replacement of HSV-1 Us3 by HSV-2 Us3 changed the phosphorylation status of UL31 and UL34, which are critical viral regulators of nuclear egress. It also caused aberrant localization of these viral proteins and aberrant accumulation of primary enveloped virions in membranous vesicle structures adjacent to the nuclear membrane, and it reduced viral cell-cell spread in cell cultures and pathogenesis in mice. These results clearly demonstrated biological differences between HSV-1 Us3 and HSV-2 Us3, especially in regulation of viral nuclear egress and phosphorylation of viral regulators critical for this process. Our study also suggested that the regulatory role(s) of HSV-1 Us3, which was not carried out by HSV-2 Us3, was important for HSV-1 cell-cell spread and pathogenesis in vivo. A previous study comparing the phenotypes of HSV-1 and HSV-2 suggested that the HSV-2 Us3 kinase lacked some of the functions of HSV-1 Us3 kinase. The difference between HSV-1 and HSV-2 Us3 kinases appeared to be due to the fact that some Us3 phosphorylation sites in HSV-1 proteins are not conserved in the corresponding HSV-2 proteins. Therefore, we generated recombinant HSV-1 strains YK781 (Us3-chimera) with HSV-2 Us3

  1. Transcriptional Analysis of Essential Genes of the Escherichia coli Fatty Acid Biosynthesis Gene Cluster by Functional Replacement with the Analogous Salmonella typhimurium Gene Cluster

    PubMed Central

    Zhang, Yan; Cronan, John E.

    1998-01-01

    The genes encoding several key fatty acid biosynthetic enzymes (called the fab cluster) are clustered in the order plsX-fabH-fabD-fabG-acpP-fabF at min 24 of the Escherichia coli chromosome. A difficulty in analysis of the fab cluster by the polar allele duplication approach (Y. Zhang and J. E. Cronan, Jr., J. Bacteriol. 178:3614–3620, 1996) is that several of these genes are essential for the growth of E. coli. We overcame this complication by use of the fab gene cluster of Salmonella typhimurium, a close relative of E. coli, to provide functions necessary for growth. The S. typhimurium fab cluster was isolated by complementation of an E. coli fabD mutant and was found to encode proteins with >94% homology to those of E. coli. However, the S. typhimurium sequences cannot recombine with the E. coli sequences required to direct polar allele duplication via homologous recombination. Using this approach, we found that although approximately 60% of the plsX transcripts initiate at promoters located far upstream and include the upstream rpmF ribosomal protein gene, a promoter located upstream of the plsX coding sequence (probably within the upstream gene, rpmF) is sufficient for normal growth. We have also found that the fabG gene is obligatorily cotranscribed with upstream genes. Insertion of a transcription terminator cassette (Ω-Cm cassette) between the fabD and fabG genes of the E. coli chromosome abolished fabG transcription and blocked cell growth, thus providing the first indication that fabG is an essential gene. Insertion of the Ω-Cm cassette between fabH and fabD caused greatly decreased transcription of the fabD and fabG genes and slower cellular growth, indicating that fabD has only a weak promoter(s). PMID:9642179

  2. Ankle replacement

    MedlinePlus

    ... is surgery to replace the damaged bone and cartilage in the ankle joint. Artificial joint parts (prosthetics) ... Your surgeon will remove the damaged bone and cartilage. Your surgeon will replace the damaged part of: ...

  3. Hip Replacement

    MedlinePlus

    ... replacement is an operation in which a damaged hip joint is removed and replaced with an artificial joint. ... are many medical conditions that can damage the hip joint. (Watch the video to learn about what goes ...

  4. Predominance of a Versatile-Peroxidase-Encoding Gene, mnp4, as Demonstrated by Gene Replacement via a Gene Targeting System for Pleurotus ostreatus

    PubMed Central

    Salame, Tomer M.; Knop, Doriv; Tal, Dana; Levinson, Dana; Yarden, Oded

    2012-01-01

    Pleurotus ostreatus (the oyster mushroom) and other white rot filamentous basidiomycetes are key players in the global carbon cycle. P. ostreatus is also a commercially important edible fungus with medicinal properties and is important for biotechnological and environmental applications. Efficient gene targeting via homologous recombination (HR) is a fundamental tool for facilitating comprehensive gene function studies. Since the natural HR frequency in Pleurotus transformations is low (2.3%), transformed DNA is predominantly integrated ectopically. To overcome this limitation, a general gene targeting system was developed by producing a P. ostreatus PC9 homokaryon Δku80 strain, using carboxin resistance complemented by the development of a protocol for hygromycin B resistance protoplast-based DNA transformation and homokaryon isolation. The Δku80 strain exhibited exclusive (100%) HR in the integration of transforming DNA, providing a high efficiency of gene targeting. Furthermore, the Δku80 strains produced showed a phenotype similar to that of the wild-type PC9 strain, with similar growth fitness, ligninolytic functionality, and capability of mating with the incompatible strain PC15 to produce a dikaryon which retained its resistance to the corresponding selection and was capable of producing typical fruiting bodies. The applicability of this system is demonstrated by inactivation of the versatile peroxidase (VP) encoded by mnp4. This enzyme is part of the ligninolytic system of P. ostreatus, being one of the nine members of the manganese-peroxidase (MnP) gene family, and is the predominantly expressed VP in Mn2+-deficient media. mnp4 inactivation provided a direct proof that mnp4 encodes a key VP responsible for the Mn2+-dependent and Mn2+-independent peroxidase activity under Mn2+-deficient culture conditions. PMID:22636004

  5. Predominance of a versatile-peroxidase-encoding gene, mnp4, as demonstrated by gene replacement via a gene targeting system for Pleurotus ostreatus.

    PubMed

    Salame, Tomer M; Knop, Doriv; Tal, Dana; Levinson, Dana; Yarden, Oded; Hadar, Yitzhak

    2012-08-01

    Pleurotus ostreatus (the oyster mushroom) and other white rot filamentous basidiomycetes are key players in the global carbon cycle. P. ostreatus is also a commercially important edible fungus with medicinal properties and is important for biotechnological and environmental applications. Efficient gene targeting via homologous recombination (HR) is a fundamental tool for facilitating comprehensive gene function studies. Since the natural HR frequency in Pleurotus transformations is low (2.3%), transformed DNA is predominantly integrated ectopically. To overcome this limitation, a general gene targeting system was developed by producing a P. ostreatus PC9 homokaryon Δku80 strain, using carboxin resistance complemented by the development of a protocol for hygromycin B resistance protoplast-based DNA transformation and homokaryon isolation. The Δku80 strain exhibited exclusive (100%) HR in the integration of transforming DNA, providing a high efficiency of gene targeting. Furthermore, the Δku80 strains produced showed a phenotype similar to that of the wild-type PC9 strain, with similar growth fitness, ligninolytic functionality, and capability of mating with the incompatible strain PC15 to produce a dikaryon which retained its resistance to the corresponding selection and was capable of producing typical fruiting bodies. The applicability of this system is demonstrated by inactivation of the versatile peroxidase (VP) encoded by mnp4. This enzyme is part of the ligninolytic system of P. ostreatus, being one of the nine members of the manganese-peroxidase (MnP) gene family, and is the predominantly expressed VP in Mn(2+)-deficient media. mnp4 inactivation provided a direct proof that mnp4 encodes a key VP responsible for the Mn(2+)-dependent and Mn(2+)-independent peroxidase activity under Mn(2+)-deficient culture conditions.

  6. Defective B cell tolerance in adenosine deaminase deficiency is corrected by gene therapy.

    PubMed

    Sauer, Aisha V; Morbach, Henner; Brigida, Immacolata; Ng, Yen-Shing; Aiuti, Alessandro; Meffre, Eric

    2012-06-01

    Adenosine deaminase (ADA) gene defects are among the most common causes of SCID. Restoration of purine metabolism and immune functions can be achieved by enzyme replacement therapy, or more effectively by bone marrow transplant or HSC gene therapy (HSC-GT). However, autoimmune complications and autoantibody production, including anti-nuclear antibodies (ANAs), frequently occur in ADA-SCID patients after treatment. To assess whether ADA deficiency affects the establishment of B cell tolerance, we tested the reactivity of recombinant antibodies isolated from single B cells of ADA-SCID patients before and after HSC-GT. We found that before HSC-GT, new emigrant/transitional and mature naive B cells from ADA-SCID patients contained more autoreactive and ANA-expressing clones, indicative of defective central and peripheral B cell tolerance checkpoints. We further observed impaired B cell receptor (BCR) and TLR functions in B cells after ADA inhibition, which may underlie the defects in B cell tolerance. Strikingly, after HSC-GT, ADA-SCID patients displayed quasi-normal early B cell tolerance checkpoints, as evidenced by restored removal of developing autoreactive and ANA-expressing B cells. Hence, ADA plays an essential role in controlling autoreactive B cell counterselection by regulating BCR and TLR functions.

  7. Arsenic resistance in Halobacterium sp. strain NRC-1 examined by using an improved gene knockout system.

    PubMed

    Wang, Gejiao; Kennedy, Sean P; Fasiludeen, Sabeena; Rensing, Christopher; DasSarma, Shiladitya

    2004-05-01

    The genome sequence of Halobacterium sp. strain NRC-1 encodes genes homologous to those responsible for conferring resistance to arsenic. These genes occur on both the large extrachromosomal replicon pNRC100 (arsADRC and arsR2M) and on the chromosome (arsB). We studied the role of these ars genes in arsenic resistance genetically by construction of gene knockouts. Deletion of the arsADRC gene cluster in a Halobacterium NRC-1 Deltaura3 strain resulted in increased sensitivity to arsenite and antimonite but not arsenate. In contrast, knockout of the chromosomal arsB gene did not show significantly increased sensitivity to arsenite or arsenate. We also found that knockout of the arsM gene produced sensitivity to arsenite, suggesting a second novel mechanism of arsenic resistance involving a putative arsenite(III)-methyltransferase. These results indicate that Halobacterium sp. strain NRC-1 contains an arsenite and antimonite extrusion system with significant differences from bacterial counterparts. Deletion analysis was facilitated by an improved method for gene knockouts/replacements in Halobacterium that relies on both selection and counterselection of ura3 using a uracil dropout medium and 5-fluoroorotic acid. The arsenite and antimonite resistance elements were shown to be regulated, with resistance to arsenic in the wild type inducible by exposure to a sublethal concentration of the metal. Northern hybridization and reverse transcription-PCR analyses showed that arsA, arsD, arsR, arsM, arsC, and arsB, but not arsR2, are inducible by arsenite and antimonite. We discuss novel aspects of arsenic resistance in this halophilic archaeon and technical improvements in our capability for gene knockouts in the genome.

  8. Arsenic Resistance in Halobacterium sp. Strain NRC-1 Examined by Using an Improved Gene Knockout System

    PubMed Central

    Wang, Gejiao; Kennedy, Sean P.; Fasiludeen, Sabeena; Rensing, Christopher; DasSarma, Shiladitya

    2004-01-01

    The genome sequence of Halobacterium sp. strain NRC-1 encodes genes homologous to those responsible for conferring resistance to arsenic. These genes occur on both the large extrachromosomal replicon pNRC100 (arsADRC and arsR2M) and on the chromosome (arsB). We studied the role of these ars genes in arsenic resistance genetically by construction of gene knockouts. Deletion of the arsADRC gene cluster in a Halobacterium NRC-1 Δura3 strain resulted in increased sensitivity to arsenite and antimonite but not arsenate. In contrast, knockout of the chromosomal arsB gene did not show significantly increased sensitivity to arsenite or arsenate. We also found that knockout of the arsM gene produced sensitivity to arsenite, suggesting a second novel mechanism of arsenic resistance involving a putative arsenite(III)-methyltransferase. These results indicate that Halobacterium sp. strain NRC-1 contains an arsenite and antimonite extrusion system with significant differences from bacterial counterparts. Deletion analysis was facilitated by an improved method for gene knockouts/replacements in Halobacterium that relies on both selection and counterselection of ura3 using a uracil dropout medium and 5-fluoroorotic acid. The arsenite and antimonite resistance elements were shown to be regulated, with resistance to arsenic in the wild type inducible by exposure to a sublethal concentration of the metal. Northern hybridization and reverse transcription-PCR analyses showed that arsA, arsD, arsR, arsM, arsC, and arsB, but not arsR2, are inducible by arsenite and antimonite. We discuss novel aspects of arsenic resistance in this halophilic archaeon and technical improvements in our capability for gene knockouts in the genome. PMID:15126481

  9. Androgen receptor gene CAG repeat polymorphism independently influences recovery of male sexual function after testosterone replacement therapy in postsurgical hypogonadotropic hypogonadism.

    PubMed

    Tirabassi, Giacomo; Delli Muti, Nicola; Corona, Giovanni; Maggi, Mario; Balercia, Giancarlo

    2014-05-01

    Few and contradictory studies have evaluated the possible influence of androgen receptor (AR) gene CAG repeat polymorphism on male sexual function. In this study we evaluated the role of AR gene CAG repeat polymorphism in the recovery of sexual function after testosterone replacement therapy (TRT) in men affected by postsurgical hypogonadotropic hypogonadism, a condition which is often associated with hypopituitarism and in which the sexual benefits of TRT must be distinguished from those of pituitary-function replacement therapies. Fifteen men affected by postsurgical hypogonadotropic hypogonadism were retrospectively assessed before and after TRT. Main outcome measures included sexual parameters as assessed by the International Index of Erectile Function questionnaire, levels of pituitary dependent hormones (total testosterone, free T3, free T4, cortisol, insulin-like growth factor-1 [IGF-1], prolactin), and results of genetic analysis (AR gene CAG repeat number). Plasma concentrations of free T3, free T4, cortisol, and prolactin did not vary significantly between the two phases, while testosterone and IGF-1 increased significantly after TRT. A significant improvement in all sexual parameters studied was found. The number of CAG triplets was negatively and significantly correlated with changes in all the sexual parameters, while opposite correlations were found between changes in sexual parameters and changes in testosterone levels; no correlation of change in IGF1 with change in sexual parameters was reported. On multiple linear regression analysis, after correction for changes in testosterone, nearly all the associations between the number of CAG triplets and changes in sexual parameters were confirmed. Shorter length AR gene CAG repeat number is associated with the recovery of sexual function after TRT in postsurgical male hypogonadotropic hypogonadism, independently of the effects of concomitant pituitary-replacement therapies. © 2014 International Society

  10. Codon-optimized Fluorescent mTFP and mCherry for Microscopic Visualization and Genetic Counterselection of Streptococci and Enterococci

    PubMed Central

    Vickerman, M. Margaret; Mansfield, Jillian M.; Zhu, Min; Walters, Katherine S.; Banas, Jeffrey A.

    2015-01-01

    Despite the powerful potential of fluorescent proteins for labeling bacteria, their use has been limited in multispecies oral biofilm models. Fermentative metabolism by streptococcal species that initiate biofilm colonization results in an acidic, reduced microenvironment that may limit the activities of some fluorescent proteins which are influenced by pH and oxygen availability. The need to reliably distinguish morphologically similar strains within biofilms was the impetus for this work. Teal fluorescent protein (mTFP1) and red fluorescent protein (mCherry) were chosen because their fluorescent properties made them promising candidates. Since tRNA availability has been implicated in efficient translation of sufficient quantities of protein for maximum fluorescence, a streptococcal codon optimization approach was used. DNA was synthesized to encode either protein using codons most frequently used in streptococci; each coding region was preceded by an engineered ribosomal binding site and restriction sites for cloning a promoter. Plasmids carrying this synthesized DNA under control of the Streptococcus mutans lactate dehydrogenase promoter conferred fluorescence to nine representative streptococcal and two Enterococcus faecalis strains. Further characterization in Streptococcus gordonii showed that mTFP1 and mCherry expression could be detected in cells grown planktonically, in biofilms, or in colonies on agar when expressed on an extrachromosomal plasmid or in single copy integrated into the chromosome. This latter property facilitated counterselection of chromosomal mutations demonstrating value for bacterial strain construction. Fluorescent and non-fluorescent bacteria were distinguishable at acidic pH. These codon-optimized versions of mTFP1 and mCherry have promising potential for use in multiple experimental applications. PMID:26122309

  11. Hormone Replacement Therapy Associated White Blood Cell DNA Methylation and Gene Expression are Associated With Within-Pair Differences of Body Adiposity and Bone Mass.

    PubMed

    Bahl, Aileen; Pöllänen, Eija; Ismail, Khadeeja; Sipilä, Sarianna; Mikkola, Tuija M; Berglund, Eva; Lindqvist, Carl Mårten; Syvänen, Ann-Christine; Rantanen, Taina; Kaprio, Jaakko; Kovanen, Vuokko; Ollikainen, Miina

    2015-12-01

    The loss of estrogen during menopause causes changes in the female body, with wide-ranging effects on health. Estrogen-containing hormone replacement therapy (HRT) leads to a relief of typical menopausal symptoms, benefits bone and muscle health, and is associated with tissue-specific gene expression profiles. As gene expression is controlled by epigenetic factors (including DNA methylation), many of which are environmentally sensitive, it is plausible that at least part of the HRT-associated gene expression is due to changes in DNA methylation profile. We investigated genome-wide DNA methylation and gene expression patterns of white blood cells (WBCs) and their associations with body composition, including muscle and bone measures of monozygotic (MZ) female twin pairs discordant for HRT. We identified 7,855 nominally significant differentially methylated regions (DMRs) associated with 4,044 genes. Of the genes with DMRs, five (ACBA1, CCL5, FASLG, PPP2R2B, and UHRF1) were also differentially expressed. All have been previously associated with HRT or estrogenic regulation, but not with HRT-associated DNA methylation. All five genes were associated with bone mineral content (BMC), and ABCA1, FASLG, and UHRF1 were also associated with body adiposity. Our study is the first to show that HRT associates with genome-wide DNA methylation alterations in WBCs. Moreover, we show that five differentially expressed genes with DMRs associate with clinical measures, including body fat percentage, lean body mass, bone mass, and blood lipids. Our results indicate that at least part of the known beneficial HRT effects on body composition and bone mass may be regulated by DNA methylation associated alterations in gene expression in circulating WBCs.

  12. Shoulder replacement

    MedlinePlus

    ... the opening at the end of the shoulder blade, called the socket. This type of joint allows ... head. The socket part (glenoid) of your shoulder blade will be replaced with a smooth plastic shell ( ...

  13. Cytosine deaminase as a negative selection marker for gene disruption and replacement in the genus Streptomyces and other actinobacteria.

    PubMed

    Dubeau, Marie-Pierre; Ghinet, Mariana Gabriela; Jacques, Pierre-Etienne; Clermont, Nancy; Beaulieu, Carole; Brzezinski, Ryszard

    2009-02-01

    We developed a novel negative selection system for actinobacteria based on cytosine deaminase (CodA). We constructed vectors that include a synthetic gene encoding the CodA protein from Escherichia coli optimized for expression in Streptomyces species. Gene disruption and the introduction of an unmarked in-frame deletion were successfully achieved with these vectors.

  14. SegH and Hef: two novel homing endonucleases whose genes replace the mobC and mobE genes in several T4-related phages

    PubMed Central

    Sandegren, Linus; Nord, David; Sjöberg, Britt-Marie

    2005-01-01

    T4 contains two groups of genes with similarity to homing endonucleases, the seg-genes (similarity to endonucleases encoded by group I introns) containing GIY-YIG motifs and the mob-genes (similarity to mobile endonucleases) containing H-N-H motifs. The four seg-genes characterized to date encode homing endonucleases with cleavage sites close to their respective gene loci while none of the mob-genes have been shown to cleave DNA. Of 18 phages screened, only T4 was found to have mobC while mobE genes were found in five additional phages. Interestingly, three phages encoded a seg-like gene (hereby called segH) with a GIY-YIG motif in place of mobC. An additional phage has an unrelated gene called hef (homing endonuclease-like function) in place of the mobE gene. The gene products of both novel genes displayed homing endonuclease activity with cleavage site specificity close to their respective genes. In contrast to intron encoded homing endonucleases, both SegH and Hef can cleave their own DNA as well as DNA from phages without the genes. Both segH and mobE (and most likely hef) can home between phages in mixed infections. We discuss why it might be a selective advantage for phage freestanding homing endonucleases to cleave both HEG-containing and HEG-less genomes. PMID:16257983

  15. SegH and Hef: two novel homing endonucleases whose genes replace the mobC and mobE genes in several T4-related phages.

    PubMed

    Sandegren, Linus; Nord, David; Sjöberg, Britt-Marie

    2005-01-01

    T4 contains two groups of genes with similarity to homing endonucleases, the seg-genes (similarity to endonucleases encoded by group I introns) containing GIY-YIG motifs and the mob-genes (similarity to mobile endonucleases) containing H-N-H motifs. The four seg-genes characterized to date encode homing endonucleases with cleavage sites close to their respective gene loci while none of the mob-genes have been shown to cleave DNA. Of 18 phages screened, only T4 was found to have mobC while mobE genes were found in five additional phages. Interestingly, three phages encoded a seg-like gene (hereby called segH) with a GIY-YIG motif in place of mobC. An additional phage has an unrelated gene called hef (homing endonuclease-like function) in place of the mobE gene. The gene products of both novel genes displayed homing endonuclease activity with cleavage site specificity close to their respective genes. In contrast to intron encoded homing endonucleases, both SegH and Hef can cleave their own DNA as well as DNA from phages without the genes. Both segH and mobE (and most likely hef) can home between phages in mixed infections. We discuss why it might be a selective advantage for phage freestanding homing endonucleases to cleave both HEG-containing and HEG-less genomes.

  16. Type of Renal Replacement Therapy (Hemodialysis versus Peritoneal Dialysis) Does Not Affect Cytokine Gene Expression or Clinical Parameters of Renal Transplant Candidates.

    PubMed

    Kamińska, Dorota; Kościelska-Kasprzak, Katarzyna; Chudoba, Paweł; Mazanowska, Oktawia; Banasik, Mirosław; Żabinska, Marcelina; Boratyńska, Maria; Lepiesza, Agnieszka; Korta, Krzysztof; Gomółkiewicz, Agnieszka; Dzięgiel, Piotr; Klinger, Marian

    2015-01-01

    Patients with renal failure suffer from immune disturbances, caused by uremic toxins and influenced by dialysis treatment. The aim of the present study was to reveal whether type of dialysis modality (hemodialysis, HD, versus peritoneal dialysis, PD) differentially affects the immunocompetence, particularly the expression of genes involved in the immune response. 87 renal transplant candidates (66 HD, 21 PD) were included in the study. The peripheral blood RNA samples were obtained with the PAXgene Blood system just before transplantation. The gene expression of CASP3, FAS, TP53, FOXP3, IFNG, IL2, IL6, IL8, IL10, IL17, IL18, LCN2, TGFB1, and TNF was assessed with real-time PCR on custom-designed low density arrays (TaqMan). Gene expression data were analyzed in relation to pretransplant clinical parameters. The mean expression of examined genes showed no significant differences between PD and HD with the exception of FAS, expression of which was 30% higher in PD patients compared to the HD group. There was nonsignificantly higher expression of proinflammatory cytokines in the PD group. The clinical inflammatory parameters (CRP, albumin, cholesterol, and hemoglobin levels) did not differ between the groups. Type of renal replacement therapy exerts no differential effect on cytokine gene expression or inflammatory clinical parameters.

  17. Glyphosate Resistance as a Novel Select-Agent-Compliant, Non-Antibiotic-Selectable Marker in Chromosomal Mutagenesis of the Essential Genes asd and dapB of Burkholderia pseudomallei▿

    PubMed Central

    Norris, Michael H.; Kang, Yun; Lu, Diana; Wilcox, Bruce A.; Hoang, Tung T.

    2009-01-01

    Genetic manipulation of the category B select agents Burkholderia pseudomallei and Burkholderia mallei has been stifled due to the lack of compliant selectable markers. Hence, there is a need for additional select-agent-compliant selectable markers. We engineered a selectable marker based on the gat gene (encoding glyphosate acetyltransferase), which confers resistance to the common herbicide glyphosate (GS). To show the ability of GS to inhibit bacterial growth, we determined the effective concentrations of GS against Escherichia coli and several Burkholderia species. Plasmids based on gat, flanked by unique flip recombination target (FRT) sequences, were constructed for allelic-replacement. Both allelic-replacement approaches, one using the counterselectable marker pheS and the gat-FRT cassette and one using the DNA incubation method with the gat-FRT cassette, were successfully utilized to create deletions in the asd and dapB genes of wild-type B. pseudomallei strains. The asd and dapB genes encode an aspartate-semialdehyde dehydrogenase (BPSS1704, chromosome 2) and dihydrodipicolinate reductase (BPSL2941, chromosome 1), respectively. Mutants unable to grow on media without diaminopimelate (DAP) and other amino acids of this pathway were PCR verified. These mutants displayed cellular morphologies consistent with the inability to cross-link peptidoglycan in the absence of DAP. The B. pseudomallei 1026b Δasd::gat-FRT mutant was complemented with the B. pseudomallei asd gene on a site-specific transposon, mini-Tn7-bar, by selecting for the bar gene (encoding bialaphos/PPT resistance) with PPT. We conclude that the gat gene is one of very few appropriate, effective, and beneficial compliant markers available for Burkholderia select-agent species. Together with the bar gene, the gat cassette will facilitate various genetic manipulations of Burkholderia select-agent species. PMID:19648360

  18. Esophageal replacement.

    PubMed

    Kunisaki, Shaun M; Coran, Arnold G

    2017-04-01

    This article focuses on esophageal replacement as a surgical option for pediatric patients with end-stage esophageal disease. While it is obvious that the patient׳s own esophagus is the best esophagus, persisting with attempts to retain a native esophagus with no function and at all costs are futile and usually detrimental to the overall well-being of the child. In such cases, the esophagus should be abandoned, and the appropriate esophageal replacement is chosen for definitive reconstruction. We review the various types of conduits used for esophageal replacement and discuss the unique advantages and disadvantages that are relevant for clinical decision-making. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Improvement in reproductive parameters in hypogonadal female mice by regulated gene replacement therapy in the central nervous system.

    PubMed

    Jeong, K-H; Bakowska, J C; Song, I O; Fu, N; Breakefield, X O; Kaiser, U B

    2007-07-01

    One of the challenges of gene targeting is to achieve regulated transgene expression in specific target cells. The hypogonadal (hpg) mice are genetically deficient in hypothalamic gonadotropin-releasing hormone (GnRH) production due to a deletion in the GnRH gene, resulting in hypogonadotropic hypogonadism. Here we show an improvement in reproductive parameters of adult female homozygous hpg mice by direct infusion into the hypothalamic preoptic area (POA) of a herpes simplex virus (HSV)-based amplicon vector containing a 13.5 kb genomic fragment encoding the GnRH gene together with its cognate promoter and regulatory elements. Following vector injection, GnRH-expressing neurons were detected in the POA, and pituitary and plasma gonadotropin levels as well as ovarian and uterine weights increased. In addition, a subset of injected hpg mice demonstrated cyclic estrous changes, consistent with regulated control of GnRH production. Administration of kisspeptin-10 resulted in an increase in plasma luteinizing hormone levels, further supporting appropriate regulation of the introduced GnRH transgene. These findings indicate that delivery of the GnRH gene resulted in selective neuronal expression of GnRH and regulated hypothalamic GnRH release. To our knowledge, this is the first example of the correct targeting of a gene under its cognate promoter to neurons resulting in selective and regulated synthesis of a biologically active peptide, and thus may have a wide range of applications in the treatment of human disorders.

  20. Are genes destiny? Have adenine, cytosine, guanine and thymine replaced Lachesis, Clotho and Atropos as the weavers of our fate?

    PubMed Central

    EISENBERG, LEON

    2005-01-01

    It is as futile to ask how much of the phenotype of an organism is due to nature and how much to its nurture as it is to determine how much of the area of a rectangle is due to its length and how much to its height. Phenotype and area are joint products. The spectacular success of genomics, unfortunately, threatens to re-awaken belief in genes as the principal determinants of human behavior. This paper develops the thesis that gene expression is modified by environmental inputs and that the impact of the environment on a given organism is modified by its genome. Genes set the boundaries of the possible; environments parse out the actual. PMID:16633494

  1. Development of a markerless gene replacement system for Acidithiobacillus ferrooxidans and construction of a pfkB mutant.

    PubMed

    Wang, Huiyan; Liu, Xiangmei; Liu, Shuangshuang; Yu, Yangyang; Lin, Jianqun; Lin, Jianqiang; Pang, Xin; Zhao, Jian

    2012-03-01

    The extremely acidophilic, chemolithoautotrophic Acidithiobacillus ferrooxidans is an important bioleaching bacterium of great value in the metallurgical industry and environmental protection. In this report, a mutagenesis system based on the homing endonuclease I-SceI was developed to produce targeted, unmarked gene deletions in the strain A. ferrooxidans ATCC 23270. A targeted phosphofructokinase (PFK) gene (pfkB) mutant of A. ferrooxidans ATCC 23270 was constructed by homologous recombination and identified by PCR with specific primers as well as Southern blot analysis. This potential pfkB gene (AFE_1807) was also characterized by expression in PFK-deficient Escherichia coli cells, and heteroexpression of the PFKB protein demonstrated that it had functional PFK activity, though it was significantly lower (about 800-fold) than that of phosphofructokinase-2 (PFK-B) expressed by the pfkB gene from E. coli K-12. The function of the potential PFKB protein in A. ferrooxidans was demonstrated by comparing the properties of the pfkB mutant with those of the wild type. The pfkB mutant strain displayed a relatively reduced growth capacity in S(0) medium (0.5% [wt/vol] elemental sulfur in 9K basal salts solution adjusted to pH 3.0 with H(2)SO(4)), but the mutation did not completely prevent A. ferrooxidans from assimilating exogenous glucose. The transcriptional analysis of some related genes in central carbohydrate metabolism in the wild-type and mutant strains with or without supplementation of glucose was carried out by quantitative reverse transcription-PCR. This report suggests that the markerless mutagenesis strategy could serve as a model for functional studies of other genes of interest from A. ferrooxidans and multiple mutations could be made in a single A. ferrooxidans strain.

  2. Integration and gene replacement in the Lactococcus lactis lac operon: induction of a cryptic phospho-beta-glucosidase in LacG-deficient strains.

    PubMed Central

    Simons, G; Nijhuis, M; de Vos, W M

    1993-01-01

    Insertions, replacement mutations, and deletions were introduced via single or double crossover recombination into the lacE (enzyme IIlac) and lacG (phospho-beta-galactosidase) genes of the Lactococcus lactis chromosomal lacABCDFEGX operon. LacG production was abolished in strains missing the lacG gene or carrying multicopy insertions in the lacE gene that affected expression of the lacG gene. However, these LacG-deficient strains could still ferment lactose slowly and were found to contain an enzymatic activity that hydrolyzed the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside phosphate. Induction of this phospho-beta-glycohydrolase activity coincided with the appearance of a new 55-kDa protein cross-reacting with anti-LacG antibodies that had a size similar to that of LacG but a higher isoelectric point (pI 5.2) and was not found in wild-type cells during growth on lactose. Since the phospho-beta-glycohydrolase activity and this protein with a pI of 5.2 were highly induced in both mutant and wild-type cells during growth on cellobiose that is likely to be transported via a phosphoenolpyruvate-dependent phosphotransferase system, we propose that this induced activity is a phospho-beta-glucosidase that also hydrolyzes lactose-6-phosphate. Images PMID:8349556

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

    PubMed

    Schaeffer, Scott M; Nakata, Paul A

    2015-11-01

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

  4. Replacement of Imu-Cmu intron by NeoR gene alters Imu germ-line expression but has no effect on V(D)J recombination.

    PubMed

    Haddad, Dania; Dougier, Hei-Lanne; Laviolette, Nathalie; Puget, Nadine; Khamlichi, Ahmed Amine

    2010-02-01

    The NeoR gene has often been used to unravel the mechanisms underlying long-range interactions between promoters and enhancers during V(D)J assembly and class switch recombination (CSR) in the immunoglobulin heavy chain (IgH) locus. This approach led to the notion that CSR is regulated through competition of germ-line (GL) promoters for activities displayed by the 3' regulatory region (3'RR). This polarized long-range effect of the 3'RR is disturbed upon insertion of NeoR gene in the IgH constant (C(H)) region, where only GL transcription derived from upstream GL promoters is impaired. In the context of V(D)J recombination, replacement of Emu enhancer or Emu core enhancer (cEmu) by NeoR gene fully blocked V(D)J recombination and mu0 GL transcription which originates 5' of DQ52 and severely diminished Imu GL transcription derived from Emu/Imu promoter, suggesting a critical role for cEmu in the regulation of V(D)J recombination and of mu0 and Imu expression. Here we focus on the effect of NeoR gene on mu0 and Imu GL transcription in a mouse line in which the Imu-Cmu intron was replaced by a NeoR gene in the sense-orientation. B cell development was characterized by a marked but incomplete block at the pro-B cell stage. However, V(D)J recombination was unaffected in sorted pro-B and pre-B cells excluding an interference with the accessibility control function of Emu. mu0 GL transcription initiation was relatively normal but the maturation step seemed to be affected most likely through premature termination at NeoR polyadenylation sites. In contrast, Imu transcription initiation was impaired suggesting an interference of NeoR gene with the IgH enhancers that control Imu expression. Surprisingly, in stark contrast with the NeoR effect in the C(H) region, LPS-induced NeoR expression restored Imu transcript levels to normal. The data suggest that Emu enhancer may be the master control element that counteracts the down-regulatory "Neo effect" on Imu expression upon LPS

  5. Protein replacement therapy and gene transfer in canine models of hemophilia A, hemophilia B, von willebrand disease, and factor VII deficiency.

    PubMed

    Nichols, Timothy C; Dillow, Aaron M; Franck, Helen W G; Merricks, Elizabeth P; Raymer, Robin A; Bellinger, Dwight A; Arruda, Valder R; High, Katherine A

    2009-01-01

    Dogs with hemophilia A, hemophilia B, von Willebrand disease (VWD), and factor VII deficiency faithfully recapitulate the severe bleeding phenotype that occurs in humans with these disorders. The first rational approach to diagnosing these bleeding disorders became possible with the development of reliable assays in the 1940s through research that used these dogs. For the next 60 years, treatment consisted of replacement of the associated missing or dysfunctional protein, first with plasma-derived products and subsequently with recombinant products. Research has consistently shown that replacement products that are safe and efficacious in these dogs prove to be safe and efficacious in humans. But these highly effective products require repeated administration and are limited in supply and expensive; in addition, plasma-derived products have transmitted bloodborne pathogens. Recombinant proteins have all but eliminated inadvertent transmission of bloodborne pathogens, but the other limitations persist. Thus, gene therapy is an attractive alternative strategy in these monogenic disorders and has been actively pursued since the early 1990s. To date, several modalities of gene transfer in canine hemophilia have proven to be safe, produced easily detectable levels of transgene products in plasma that have persisted for years in association with reduced bleeding, and correctly predicted the vector dose required in a human hemophilia B liver-based trial. Very recently, however, researchers have identified an immune response to adeno-associated viral gene transfer vector capsid proteins in a human liver-based trial that was not present in preclinical testing in rodents, dogs, or nonhuman primates. This article provides a review of the strengths and limitations of canine hemophilia, VWD, and factor VII deficiency models and of their historical and current role in the development of improved therapy for humans with these inherited bleeding disorders.

  6. Polymorphisms of the GR and HSD11B1 genes influence body mass index and weight gain during hormone replacement treatment in patients with Addison's disease.

    PubMed

    Molnár, Ágnes; Kövesdi, Annamária; Szücs, Nikolette; Tóth, Miklós; Igaz, Péter; Rácz, Károly; Patócs, Attila

    2016-08-01

    Glucocorticoid substitution is essential in patients with chronic primary adrenocortical insufficiency (Addison's disease) and both over-treatment and inadequate dosage have deleterious effects. Individual sensitivity to glucocorticoids is partly genetically determined. To test the hypothesis whether the well-characterized SNPs of the GR and HSD11B1 genes may modulate the individual sensitivity to exogenous glucocorticoids and may influence clinical and/or laboratory parameters and the glucocorticoid substitution dosage in patients with Addison's disease. 68 patients with primary adrenocortical insufficiency were involved. Clinical and laboratory data, as well as the dosage of the hormone replacement therapy were collected. Peripheral blood DNA was isolated, and the GR and HSD11B1 SNPs were examined using allele-specific PCR or Taqman assay on Real Time PCR. The allele frequency of the GR N363S polymorphism was higher in patients compared to the control group and the disease appeared significantly earlier in patients harbouring the GR A3669G compared to noncarriers. These patients had higher ACTH level measured at the time of diagnosis. Homozygous BclI carriers had higher body mass index (BMI) and lower total hydrocortisone equivalent supplementation dose needed than heterozygous or noncarriers. The BMI and weight gain during hormone replacement therapy were also higher in carriers of the HSD11B1 rs4844880 treated with glucocorticoids other than dexamethasone. The BclI polymorphism of the GR gene and the rs4844880 of the HSD11B1 gene may contribute to weight gain and may affect the individual need of glucocorticoid substitution dose in these patients. © 2016 John Wiley & Sons Ltd.

  7. [Comparison between poly hydroxy acrylic acid and Van-clear replacing the traditional reagents to detect the cervical hTERC genes by adopting FISH technique].

    PubMed

    Chen, Z Q; Wang, Y; Mi, X J; Chen, A; Huang, H Y; Zhong, S J; Deng, W T; Liu, C F; Xu, X M; Dai, X Z

    2016-04-18

    To observe the difference of the human telomeres RNA component (hTERC) genes' amplification in the cervical tissue by applying the environment-friendly fixative poly hydroxy acrylic acid and the transparent dewaxing solution Van-clear separately or jointly to replace the traditional fixative 4% (volume fraction) neutral buffered formalin and the conventional transparent dewaxing solution xylene in the use of fluorescence in situ hybridization (FISH) for detection. In the study, 255 cases of cervical tissue specimens submitted by the Department of Gynecology in Zhongshan Boai Hosipital were collected from Mar. 2013 to Apr. 2015. Four samples were taken from the same lesion site. All the cases were divided into 4 groups and named group A, B, C, and D. Group A used 4% neutral buffered formalin fixed and xylene dewaxing to make slices. Group B used poly hydroxy acrylic fixed and xylene dewaxing to make slices. Group C used 4% neutral buffered formalin fixed and Van-clear transparent to make slices. Group D used poly hydroxy acrylic fixed and Van-clear transparent dewaxing to make slices. The amplification of hTERC genes in the four groups of cervical specimens was also detected by FISH technique. When the hTERC genes were detected by FISH method under the fluorescence microscope, it was obvious that the tissue profile and the background of group A, B, C and D were all clear. The probe was fixed in the accurate position so that the bright red or green fluorescence signals were easily found in these four groups. Compared with the positive rate of group A, there was no statistical significance in that of group B, C and D (P>0.05). At the same time, the coincidence rate of the FISH results was high, which showed that the new environment-friendly reagent had no significant difference in the detection of cervical hTERC genes by FISH technique. It is possible for the environment-friendly reagent poly hydroxy acrylic acid and Van-clear to replace 4% neutral buffered formalin

  8. Significance of Calcium Binding, Tyrosine Phosphorylation, and Lysine Trimethylation for the Essential Function of Calmodulin in Vertebrate Cells Analyzed in a Novel Gene Replacement System*

    PubMed Central

    Panina, Svetlana; Stephan, Alexander; la Cour, Jonas M.; Jacobsen, Kivin; Kallerup, Line K.; Bumbuleviciute, Rasita; Knudsen, Kristoffer V. K.; Sánchez-González, Pablo; Villalobo, Antonio; Olesen, Uffe H.; Berchtold, Martin W.

    2012-01-01

    Calmodulin (CaM) was shown to be essential for survival of lower eukaryotes by gene deletion experiments. So far, no CaM gene deletion was reported in higher eukaryotes. In vertebrates, CaM is expressed from several genes, which encode an identical protein, making it difficult to generate a model system to study the effect of CaM gene deletion. Here, we present a novel genetic system based on the chicken DT40 cell line, in which the two functional CaM genes were deleted and one allele replaced with a CaM transgene that can be artificially regulated. We show that CaM is essential for survival of vertebrate cells as they die in the absence of CaM expression. Reversal of CaM repression or ectopic expression of HA-tagged CaM rescued the cells. Cells exclusively expressing HA-CaM with impaired individual calcium binding domains as well as HA-CaM lacking the ability to be phosphorylated at residues Tyr99/Tyr138 or trimethylated at Lys115 survived and grew well. CaM mutated at both Ca2+ binding sites 3 and 4 as well as at both sites 1 and 2, but to a lesser degree, showed decreased ability to support cell growth. Cells expressing CaM with all calcium binding sites impaired died with kinetics similar to that of cells expressing no CaM. This system offers a unique opportunity to analyze CaM structure-function relationships in vivo without the use of pharmacological inhibitors and to analyze the function of wild type and mutated CaM in modulating the activity of different target systems without interference of endogenous CaM. PMID:22493455

  9. Positive association of the hepatic lipase gene polymorphism c.514C > T with estrogen replacement therapy response

    PubMed Central

    2011-01-01

    Background Hepatic lipase (HL), an enzyme present in the hepatic sinusoids, is responsible for the lipolysis of lipoproteins. Human HL contains four polymorphic sites: G-250A, T-710C, A-763G, and C-514T single-nucleotide polymorphism (SNPs). The last polymorphism is the focus of the current study. The genotypes associated with the C-514T polymorphism are CC (normal homozygous - W), CT (heterozygous - H), and TT (minor-allele homozygous - M). HL activity is significantly impaired in individuals of the TT and CT genotypes. A total of 58 post-menopausal women were studied. The subjects were hysterectomized women receiving hormone replacement therapy consisting of 0.625 mg of conjugated equine estrogen once a day. The inclusion criteria were menopause of up to three years and normal blood tests, radiographs, cervical-vaginal cytology, and densitometry. DNA was extracted from the buccal and blood cells of all 58 patients using a commercially available kit (GFX® - Amersham-Pharmacia, USA). Results Statistically significant reductions in triglycerides (t = 2.16; n = 58; p = 0.03) but not in total cholesterol (t = 0.14; n = 58; p = 0.89) were found after treatment. This group of good responders were carriers of the T allele; the CT and TT genotypes were present significantly more frequently than in the group of non-responders (p = 0.02 or p = 0.07, respectively). However, no significant difference in HDL-C (t = 0.94; n = 58; p = 0.35) or LDL-C (t = -0.83; n = 58; p = 0.41) was found in these patients. Conclusions The variation in lipid profile associated with the C-514T polymorphism is significant, and the T allele is associated with the best response to ERT. PMID:22047520

  10. Gene replacement and elimination using λRed- and FLP-based tool to re-direct carbon flux in acetogen biocatalyst during continuous CO₂/H₂ blend fermentation.

    PubMed

    Tyurin, Michael

    2013-07-01

    A time- and cost-efficient two-step gene elimination procedure was used for acetogen Clostridium sp. MT1834 capable of fermenting CO₂/H₂ blend to 245 mM acetate (p < 0.005). The first step rendered the targeted gene replacement without affecting the total genome size. We replaced the acetate pta-ack cluster with synthetic bi-functional acetaldehyde-alcohol dehydrogenase (al-adh). Replacement of pta-ack with al-adh rendered initiation of 243 mM ethanol accumulation at the expense of acetate production during CO₂/H₂ blend continuous fermentation (p < 0.005). At the second step, al-adh was eliminated to reduce the genome size. Resulting recombinants accumulated 25 mM mevalonate in fermentation broth (p < 0.005). Cell duplication time for recombinants with reduced genome size decreased by 9.5 % compared to Clostridium sp. MT1834 strain under the same fermentation conditions suggesting better cell energy pool management in the absence of the ack-pta gene cluster in the engineered biocatalyst. If the first gene elimination step was used alone for spo0A gene replacement with two copies of synthetic formate dehydrogenase in recombinants with a shortened genome, mevalonate production was replaced with 76.5 mM formate production in a single step continuous CO₂/H₂ blend fermentation (p < 0.005) with cell duplication time almost nearing that of the wild strain.

  11. Progressive myofiber loss with extensive fibro-fatty replacement in a child with mitochondrial DNA depletion syndrome and novel thymidine kinase 2 gene mutations.

    PubMed

    Collins, James; Bove, Kevin E; Dimmock, David; Morehart, Paula; Wong, Lee-Jun; Wong, Brenda

    2009-11-01

    The mitochondrial DNA depletion syndromes (MDS) are autosomal recessive disorders with a decreased mitochondrial DNA copy number. Mutations in thymidine kinase 2 (TK2) have been responsible for the myopathic form of MDS. We describe a child with congenital muscle weakness who had a progressive mitochondrial myopathy associated with extensive fibro-fatty replacement of myofibers resembling muscular dystrophy. MDS was suspected based upon findings in the initial muscle biopsy. Sequence analysis of the TK2 gene revealed two novel heterozygous mutations: the frame shift mutation, c.255_c.258delAGAA, and the heterozygous missense mutation, c.515G>A, (p.R172Q). This report extends the phenotype and genotype of TK2 defects.

  12. A DNA fragment from Xq21 replaces a deleted region containing the entire FVIII gene in a severe hemophilia A patient

    SciTech Connect

    Murru, S.; Casula, L.; Moi, P.

    1994-09-15

    In this paper the authors report the molecular characterization of a large deletion that removes the entire Factor VIII gene in a severe hemophilia A patient. Accurate DNA analysis of the breakpoint region revealed that a large DNA fragment replaced the 300-kb one, which was removed by the deletion. Pulsed-field gel electrophoresis analysis revealed that the size of the inserted fragment is about 550 kb. In situ hybridization demonstrated that part of the inserted region normally maps to Xq21 and to the tip of the short arm of the Y chromosome (Yp). In this patient this locus is present both in Xq21 and in Xq28, in addition to the Yp, being thus duplicated in the X chromosome. Sequence analysis of the 3` breakpoint suggested that an illegitimate recombination is probably the cause of this complex rearrangement. 52 refs., 7 figs.

  13. Histone gene replacement reveals a post-transcriptional role for H3K36 in maintaining metazoan transcriptome fidelity

    PubMed Central

    Meers, Michael P; Henriques, Telmo; Lavender, Christopher A; McKay, Daniel J; Strahl, Brian D; Duronio, Robert J; Adelman, Karen; Matera, A Gregory

    2017-01-01

    Histone H3 lysine 36 methylation (H3K36me) is thought to participate in a host of co-transcriptional regulatory events. To study the function of this residue independent from the enzymes that modify it, we used a ‘histone replacement’ system in Drosophila to generate a non-modifiable H3K36 lysine-to-arginine (H3K36R) mutant. We observed global dysregulation of mRNA levels in H3K36R animals that correlates with the incidence of H3K36me3. Similar to previous studies, we found that mutation of H3K36 also resulted in H4 hyperacetylation. However, neither cryptic transcription initiation, nor alternative pre-mRNA splicing, contributed to the observed changes in expression, in contrast with previously reported roles for H3K36me. Interestingly, knockdown of the RNA surveillance nuclease, Xrn1, and members of the CCR4-Not deadenylase complex, restored mRNA levels for a class of downregulated, H3K36me3-rich genes. We propose a post-transcriptional role for modification of replication-dependent H3K36 in the control of metazoan gene expression. DOI: http://dx.doi.org/10.7554/eLife.23249.001 PMID:28346137

  14. Genes and Gene Therapy

    MedlinePlus

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  15. Rapid replacement of human respiratory syncytial virus A with the ON1 genotype having 72 nucleotide duplication in G gene.

    PubMed

    Kim, You-Jin; Kim, Dae-Won; Lee, Wan-Ji; Yun, Mi-Ran; Lee, Ho Yeon; Lee, Han Saem; Jung, Hee-Dong; Kim, Kisoon

    2014-08-01

    Human respiratory syncytial virus (HRSV) is the main cause of severe respiratory illness in young children and elderly people. We investigated the genetic characteristics of the circulating HRSV subgroup A (HRSV-A) to determine the distribution of genotype ON1, which has a 72-nucleotide duplication in attachment G gene. We obtained 456 HRSV-A positive samples between October 2008 and February 2013, which were subjected to sequence analysis. The first ON1 genotype was discovered in August 2011 and 273 samples were identified as ON1 up to February 2013. The prevalence of the ON1 genotype increased rapidly from 17.4% in 2011-2012 to 94.6% in 2012-2013. The mean evolutionary rate of G protein was calculated as 3.275 × 10(-3) nucleotide substitution/site/year and several positively selected sites for amino acid substitutions were located in the predicted epitope region. This basic and important information may facilitate a better understanding of HRSV epidemiology and evolution.

  16. Targeted gene transfer into ependymal cells through intraventricular injection of AAV1 vector and long-term enzyme replacement via the CSF.

    PubMed

    Yamazaki, Yoshiyuki; Hirai, Yukihiko; Miyake, Koichi; Shimada, Takashi

    2014-07-01

    Enzyme replacement via the cerebrospinal fluid (CSF) has been shown to ameliorate neurological symptoms in model animals with neuropathic metabolic disorders. Gene therapy via the CSF offers a means to achieve a long-term sustainable supply of therapeutic proteins within the central nervous system (CNS) by setting up a continuous source of transgenic products. In the present study, a serotype 1 adeno-associated virus (AAV1) vector was injected into a lateral cerebral ventricle in adult mice to transduce the gene encoding human lysosomal enzyme arylsulfatase A (hASA) into the cells of the CNS. Widespread transduction and stable expression of hASA in the choroid plexus and ependymal cells was observed throughout the ventricles for more than 1 year after vector injection. Although humoral immunity to hASA developed after 6 weeks, which diminished the hASA levels detected in CSF from AAV1-injected mice, hASA levels in CSF were maintained for at least 12 weeks when the mice were tolerized to hASA prior of vector injection. Our results suggest that the cells lining the ventricles could potentially serve as a biological reservoir for long-term continuous secretion of lysosomal enzymes into the CSF following intracerebroventricular injection of an AAV1 vector.

  17. Nrf2-dependent gene expression is affected by the proatherogenic apoE4 genotype-studies in targeted gene replacement mice.

    PubMed

    Graeser, Anne-Christin; Boesch-Saadatmandi, Christine; Lippmann, Jana; Wagner, Anika E; Huebbe, Patricia; Storm, Niels; Höppner, Wolfgang; Wiswedel, Ingrid; Gardemann, Andreas; Minihane, Anne M; Döring, Frank; Rimbach, Gerald

    2011-10-01

    An apoE4 genotype is an important risk factor for cardiovascular and other chronic diseases. The higher cardiovascular disease risk of apoE4 carriers as compared to the apoE3 genotype has been mainly attributed to the differences in blood lipids between the two genotype subgroups. Recently, a potential protective role of the transcription factor Nrf2 in cardiovascular disease prevention has been suggested. In this study, we show that Nrf2-dependent gene expression is affected by the apoE genotype. ApoE4 vs. apoE3 mice exhibited lower hepatic Nrf2 nuclear protein levels. Furthermore, mRNA and protein levels of Nrf2 target genes including glutathione-S-transferase, heme oxygenase-1 and NAD(P)H dehydrogenase, quinone 1 were significantly lower in apoE4 as compared to apoE3 mice. Lower hepatic mRNA levels of phase II enzymes, as observed in apoE4 vs. apoE3 mice, were accompanied by higher mRNA levels of phase I enzymes including Cyp26a1 and Cyp3a16. Furthermore, miRNA-144, miRNA-125b, and miRNA-29a involved in Nrf2 signaling, inflammation, and regulation of phase I enzyme gene expression were affected by the apoE genotype. We provide first evidence that Nrf2 is differentially regulated in response to the apoE genotype.

  18. [The influence of hormonal replacement therapy on bone density in postmenopausal women depending on polymorphism of vitamin D receptor (VDR) and estrogen receptor (ER) genes].

    PubMed

    Brodowska, Agnieszka

    2003-01-01

    genes? 3. What estrogen concentration is necessary to protect bone tissue depending on the polymorphism of VDR and ER genes? The study group included 44 postmenopausal women aged 44-75 years with primary osteoporosis on cyclic HRT (hormonal replacement therapy). Two hormonal preparations were administered: Systen 50 (Jansen Cilag) transdermal system twice per week between day 1 and 21 of the cycle; Provera (Upjohn) 5 mg tablets daily between day 16 and 27 of the cycle. This therapy was supplemented with vitamin D analogue (Alphacalcidolum, Glaxo-Poznan) orally at 0.25 microg per day, calcium-enriched (1200 mg daily) diet and regular physical exercise. Patients were qualified to the study on the basis of a questionnaire. Women with secondary osteoporosis were excluded. TSH, FT3, and FT4 concentrations, as well as fasting glucose were measured. 24 h glycemia was established in women with elevated glucose levels. Polymorphism of the ER gene was studied with Xba I and Pvu II restrictases. Polymorphism of the VDR gene was studied with Bsm I restrictase. Age and BMI were recorded. Spine BMD was determined with DEXA (Dual Energy X-ray Absorptiometry (Lunar instrument) before treatment and after 12 months of HRT. Serum estradiol concentrations were measured before treatment and after 2 months of HTR. The following conclusions were drawn: 1. There is no connection between VDR and ER gene polymorphism and degree of osteoporosis before treatment. 2. XX, PP and Bb markers or X, P, B alleles are associated with a significant decrease in therapeutic efficacy. Nevertheless, satisfactory results were achieved in each woman with primary osteoporosis. 3. Estradiol concentration in serum before and during HRT does not depend on the polymorphism of VDR and ER genes.

  19. Replacement of dietary fish oil by vegetable oils affects humoral immunity and expression of pro-inflammatory cytokines genes in gilthead sea bream Sparus aurata.

    PubMed

    Montero, D; Mathlouthi, F; Tort, L; Afonso, J M; Torrecillas, S; Fernández-Vaquero, A; Negrin, D; Izquierdo, M S

    2010-12-01

    Commercial gilthead sea bream feeds are highly energetic, fish oil traditionally being the main lipid source. But the decreased fish oil production together with the increased prices of this oil encourages its substitution by vegetable oils, imposing new nutritional habits to aquaculture species. Partial replacement of fish oil by vegetable oils in diets for marine species allows good feed utilization and growth but may affect fish health, since imbalances in dietary fatty acids may alter fish immunological status. The effect of dietary oils on different aspects of fish immune system has been reported for some species, but very little is known about the effect of dietary oils on immune-related genes expression in fish. Thus, the objective of this study was to elucidate the role of dietary oils on the expression of two pro-inflammatory cytokines, Tumor Necrosis Factor-α (TNF-α) and Interleukine 1β (IL-1β) on intestine and head kidney after exposure to the bacterial pathogen Photobacterium damselae sp. piscicida. For that purpose, 5 iso-nitrogenous and iso-lipidic diets (45% crude protein, 22% crude lipid content) were formulated. Anchovy oil was the only lipid source used in the control diet (FO), but in the other diets, fish oil was totally (100%) or partially (70%) substituted by linseed (rich in n-3 fatty acids) or soybean (rich in n-6 fatty acids) (100L, 100S, 70L, 70S). Fish were fed experimental diets during 80 days and after this period were exposed to an experimental intestinal infection with the pathogen. Serum and tissue samples were obtained at pre-infection and after 1, 3 and 7 days of infection. RNA was extracted and cDNA was synthesized by reverse transcription from intestine and head kidney and the level expression of TNF-α and IL-1β were assayed by using quantitative real time PCR. The expression level of genes analysed was represented as relative value, using the comparative Ct method (2(-ΔΔCt)). Serum anti-bacterial activity was measured as

  20. Genome engineering using a synthetic gene circuit in Bacillus subtilis

    PubMed Central

    Jeong, Da-Eun; Park, Seung-Hwan; Pan, Jae-Gu; Kim, Eui-Joong; Choi, Soo-Keun

    2015-01-01

    Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac–chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications. PMID:25552415

  1. Genome engineering using a synthetic gene circuit in Bacillus subtilis.

    PubMed

    Jeong, Da-Eun; Park, Seung-Hwan; Pan, Jae-Gu; Kim, Eui-Joong; Choi, Soo-Keun

    2015-03-31

    Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac-chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications.

  2. Is the crowd better as an assistant or a replacement in ontology engineering? An exploration through the lens of the Gene Ontology.

    PubMed

    Mortensen, Jonathan M; Telis, Natalie; Hughey, Jacob J; Fan-Minogue, Hua; Van Auken, Kimberly; Dumontier, Michel; Musen, Mark A

    2016-04-01

    Biomedical ontologies contain errors. Crowdsourcing, defined as taking a job traditionally performed by a designated agent and outsourcing it to an undefined large group of people, provides scalable access to humans. Therefore, the crowd has the potential to overcome the limited accuracy and scalability found in current ontology quality assurance approaches. Crowd-based methods have identified errors in SNOMED CT, a large, clinical ontology, with an accuracy similar to that of experts, suggesting that crowdsourcing is indeed a feasible approach for identifying ontology errors. This work uses that same crowd-based methodology, as well as a panel of experts, to verify a subset of the Gene Ontology (200 relationships). Experts identified 16 errors, generally in relationships referencing acids and metals. The crowd performed poorly in identifying those errors, with an area under the receiver operating characteristic curve ranging from 0.44 to 0.73, depending on the methods configuration. However, when the crowd verified what experts considered to be easy relationships with useful definitions, they performed reasonably well. Notably, there are significantly fewer Google search results for Gene Ontology concepts than SNOMED CT concepts. This disparity may account for the difference in performance - fewer search results indicate a more difficult task for the worker. The number of Internet search results could serve as a method to assess which tasks are appropriate for the crowd. These results suggest that the crowd fits better as an expert assistant, helping experts with their verification by completing the easy tasks and allowing experts to focus on the difficult tasks, rather than an expert replacement. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. The consensus-based approach for gene/enzyme replacement therapies and crystallization strategies: the case of human alanine-glyoxylate aminotransferase.

    PubMed

    Mesa-Torres, Noel; Yunta, Cristina; Fabelo-Rosa, Israel; Gonzalez-Rubio, Juana María; Sánchez-Ruiz, José M; Salido, Eduardo; Albert, Armando; Pey, Angel L

    2014-09-15

    Protein stability is a fundamental issue in biomedical and biotechnological applications of proteins. Among these applications, gene- and enzyme-replacement strategies are promising approaches to treat inherited diseases that may benefit from protein engineering techniques, even though these beneficial effects have been largely unexplored. In the present study we apply a sequence-alignment statistics procedure (consensus-based approach) to improve the activity and stability of the human AGT (alanine-glyoxylate aminotransferase) protein, an enzyme which causes PH1 (primary hyperoxaluria type I) upon mutation. By combining only five consensus mutations, we obtain a variant (AGT-RHEAM) with largely enhanced in vitro thermal and kinetic stability, increased activity, and with no side effects on foldability and peroxisomal targeting in mammalian cells. The structure of AGT-RHEAM reveals changes at the dimer interface and improved electrostatic interactions responsible for increased kinetic stability. Consensus-based variants maintained the overall protein fold, crystallized more easily and improved the expression as soluble proteins in two different systems [AGT and CIPK24 (CBL-interacting serine/threonine-protein kinase) SOS2 (salt-overly-sensitive 2)]. Thus the consensus-based approach also emerges as a simple and generic strategy to increase the crystallization success for hard-to-get protein targets as well as to enhance protein stability and function for biomedical applications.

  4. A pharmacokinetic analysis of a novel enzyme replacement therapy with Gene-Activated human glucocerebrosidase (GA-GCB) in patients with type 1 Gaucher disease.

    PubMed

    Zimran, Ari; Loveday, Kenneth; Fratazzi, Candida; Elstein, Deborah

    2007-01-01

    To evaluate pharmacokinetics of Gene-Activated human glucocerebrosidase (GA-GCB), a novel enzyme replacement therapy, in patients with type 1 Gaucher disease. Open-label study of GA-GCB, administered as a 1-h intravenous (IV) infusion every other week was evaluated. The first three patients sequentially received one infusion each at 15 U/kg, 30 U/kg, and 60 U/kg at 2-week intervals and then continued with 60 U/kg/infusion every other week; subsequently nine more patients received GA-GCB at 60 U/kg/infusion every other week. Each patient received 20 infusions (40 weeks). Pharmacokinetic (PK) parameters reported are from blood samples collected at Weeks 1, 3, and 5 for dose-escalated patients and at Week 1 from the other nine patients. GA-GCB was rapidly cleared from the circulation and followed first-order elimination kinetics in the 12 patients who received IV infusions. Maximum serum concentration (C(max)) coincided with the end of the 1-h infusion. Both C(max) and area under the curve (AUC) were linearly proportional to dose from 15 U/kg to 60 U/kg. Elimination half-life was independent of dose; mean elimination half-life at 60 U/kg was approximately 10 min (range: 4-15 min). Mean serum clearance was 13 ml/min/kg (range: 9-20 ml/min/kg) and V(ss) (apparent volume of distribution) was approximately 18% body weight (range: 11-27% body weight). GA-GCB demonstrated linear PK parameters over clinically relevant doses (15 U/kg-60 U/kg) indicating that the dose of IV-administered GA-GCB to target tissues should also be linearly proportional to dose.

  5. BclI polymorphism of the glucocorticoid receptor gene is associated with increased bone resorption in patients on glucocorticoid replacement therapy.

    PubMed

    Koetz, Kathrin R; van Rossum, Elisabeth F C; Ventz, Manfred; Diederich, Sven; Quinkler, Marcus

    2013-06-01

    Patients with primary adrenal insufficiency (PAI) and patients with congenital adrenal hyperplasia (CAH) receive weight-adapted standard glucocorticoid replacement therapy. Clinically, some patients appear more sensitive to therapeutic administration of glucocorticoids than others. Glucocorticoid sensitivity is at least partially genetically determined by polymorphisms of the glucocorticoid receptor (GR) and might influence bone mineral density (BMD). To determine if bone turnover markers and BMD are associated with the GR gene polymorphism BclI in patients with PAI and CAH. A prospective, cross-sectional study including 74 PAI and 38 CAH patients. BMD was evaluated by DXA. Serum levels of bone turnover markers, minerals, vitamins and hormones, and urinary crosslinks were measured. Patients carrying the homozygous BclI polymorphism (GG) had significantly higher serum β-CrossLaps (0.37 ± 0.34 μg/l; P < 0.05) and urinary collagen crosslinks (NTX, 68.1 ± 32.4 nmol/g; P < 0.005) despite receiving the lowest average daily hydrocortisone dose of 9.9 ± 3.7 mg/m(2) (P < 0.05). The GG genotype occurred significantly more frequently in patients with increased NTX (OR=6.7, 95% CI = 1.78-25.38) than in patients with normal NTX. However, BMD was not significantly different between different allelic variants. No significant differences in associations of the genotypes with outcomes (or in clinical characteristics) were found between the sexes. Although the sample sizes were relatively small and the results should be interpreted with caution, this study suggests that the homozygous (GG) genotype may be associated with higher bone resorption in adult PAI and CAH patients. GG-carriers needed a lower hydrocortisone dose on average supporting the concept that this GR variant is associated with increased cortisol sensitivity. © 2012 John Wiley & Sons Ltd.

  6. Replacing the Promoter of the Murine Gene Encoding P-selectin with the Human Promoter Confers Human-like Basal and Inducible Expression in Mice*

    PubMed Central

    Liu, Zhenghui; Zhang, Nan; Shao, Bojing; Panicker, Sumith R.; Fu, Jianxin; McEver, Rodger P.

    2016-01-01

    In humans and mice, megakaryocytes/platelets and endothelial cells constitutively synthesize P-selectin and mobilize it to the plasma membrane to mediate leukocyte rolling during inflammation. TNF-α, interleukin 1β, and LPS markedly increase P-selectin mRNA in mice but decrease P-selectin mRNA in humans. Transgenic mice bearing the entire human SELP gene recapitulate basal and inducible expression of human P-selectin and reveal human-specific differences in P-selectin function. Differences in the human SELP and murine Selp promoters account for divergent expression in vitro, but their significance in vivo is not known. Here we generated knockin mice that replace the 1.4-kb proximal Selp promoter with the corresponding SELP sequence (SelpKI). SelpKI/KI mice constitutively expressed more P-selectin on platelets and more P-selectin mRNA in tissues but only slightly increased P-selectin mRNA after injection of TNF-α or LPS. Consistent with higher basal expression, leukocytes rolled more slowly on P-selectin in trauma-stimulated venules of SelpKI/KI mice. However, TNF-α did not further reduce P-selectin-dependent rolling velocities. Blunted up-regulation of P-selectin mRNA during contact hypersensitivity reduced P-selectin-dependent inflammation in SelpKI/− mice. Higher basal P-selectin in SelpKI/KI mice compensated for this defect. Therefore, divergent sequences in a short promoter mediate most of the functionally significant differences in expression of human and murine P-selectin in vivo. PMID:26631722

  7. Essential Gene Discovery in the Basidiomycete Cryptococcus neoformans for Antifungal Drug Target Prioritization

    PubMed Central

    Ianiri, Giuseppe

    2015-01-01

    ABSTRACT Fungal diseases represent a major burden to health care globally. As with other pathogenic microbes, there is a limited number of agents suitable for use in treating fungal diseases, and resistance to these agents can develop rapidly. Cryptococcus neoformans is a basidiomycete fungus that causes cryptococcosis worldwide in both immunocompromised and healthy individuals. As a basidiomycete, it diverged from other common pathogenic or model ascomycete fungi more than 500 million years ago. Here, we report C. neoformans genes that are essential for viability as identified through forward and reverse genetic approaches, using an engineered diploid strain and genetic segregation after meiosis. The forward genetic approach generated random insertional mutants in the diploid strain, the induction of meiosis and sporulation, and selection for haploid cells with counterselection of the insertion event. More than 2,500 mutants were analyzed, and transfer DNA (T-DNA) insertions in several genes required for viability were identified. The genes include those encoding the thioredoxin reductase (Trr1), a ribosome assembly factor (Rsa4), an mRNA-capping component (Cet1), and others. For targeted gene replacement, the C. neoformans homologs of 35 genes required for viability in ascomycete fungi were disrupted, meiosis and sporulation were induced, and haploid progeny were evaluated for their ability to grow on selective media. Twenty-one (60%) were found to be required for viability in C. neoformans. These genes are involved in mitochondrial translation, ergosterol biosynthesis, and RNA-related functions. The heterozygous diploid mutants were evaluated for haploinsufficiency on a number of perturbing agents and drugs, revealing phenotypes due to the loss of one copy of an essential gene in C. neoformans. This study expands the knowledge of the essential genes in fungi using a basidiomycete as a model organism. Genes that have no mammalian homologs and are essential

  8. Nicotine replacement therapy

    MedlinePlus

    ... nicotine replacement product, even for a short time. Alternative Names Smoking cessation - nicotine replacement; Tobacco - nicotine replacement therapy References American Cancer Society. Guide to quitting smoking. Last revised February ...

  9. Shoulder replacement - discharge

    MedlinePlus

    ... page: //medlineplus.gov/ency/patientinstructions/000178.htm Shoulder replacement - discharge To use the sharing features on this page, please enable JavaScript. You had shoulder replacement surgery to replace the bones of your shoulder ...

  10. Hip replacement - discharge

    MedlinePlus

    ... surgery to replace all or part of your hip joint with an artificial joint called a prosthesis. This ... You're in the Hospital You had a hip joint replacement surgery to replace all or part of ...

  11. Dietary fish oil replacement by linseed oil: Effect on growth, nutrient utilization, tissue fatty acid composition and desaturase gene expression in silver barb (Puntius gonionotus) fingerlings.

    PubMed

    Nayak, Madhusmita; Saha, Ashis; Pradhan, Avinash; Samanta, Mrinal; Giri, Shiba Shankar

    2017-03-01

    Silver barb (Puntius gonionotus) is considered a promising medium carp species for freshwater aquaculture in Asia. This study in silver barb was carried out to evaluate the effects of total or partial substitution of dietary fish oil (FO) with linseed oil (LO) on growth, nutrient utilization, whole-body composition, muscle and liver fatty acid composition. Fish (12.1±0.4g of initial body weight) were fed for 60days with five experimental iso-proteinous, iso-lipidic and iso-caloric diets in which FO (control diet) was replaced by 33.3%, 50%, 66.7% and 100% LO. Final weight, weight gain, percent weight gain, SGR decreased linearly (p<0.001) with increasing LO levels in the diets. Dietary LO substitution levels did not significantly (p>0.05) affect the feed conversion ratio (FCR), protein efficiency ratio (PER) and whole body proximate composition. Furthermore, enhanced level of LO increased α-linolenic acid (ALA; 18:3n3) and linoleic acid (LA; 18:2n6) and decreased eicosapentaenoic acid (EPA; 20:5n3) and docosahexaenoic acid (DHA; 22:6n3) in muscle and liver. To understand the molecular mechanism of long chain-polyunsaturated fatty acid (LC-PUFA) biosynthesis, we cloned and characterized the fatty acyl Δ6 desaturase (Δ6 fad) cDNA and investigated its expression in various organs/tissues following replacement of FO with LO in the diet. The full-length Δ6 fad cDNA was 2056bp encoding 444 amino acids and was widely expressed in various organs/tissues. Replacement of FO with LO increased the expression of Δ6 fad mRNA in liver, muscle and intestine but no significant difference was found in the brain. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Long-term replacement of a mutated nonfunctional CNS gene: reversal of hypothalamic diabetes insipidus using an EIAV-based lentiviral vector expressing arginine vasopressin.

    PubMed

    Bienemann, Alison S; Martin-Rendon, Enca; Cosgrave, Anna S; Glover, Colin P J; Wong, Liang-Fong; Kingsman, Susan M; Mitrophanous, Kyriacos A; Mazarakis, Nicholas D; Uney, James B

    2003-05-01

    Due to the complexity of brain function and the difficulty in monitoring alterations in neuronal gene expression, the potential of lentiviral gene therapy vectors to treat disorders of the CNS has been difficult to fully assess. In this study, we have assessed the utility of a third-generation equine infectious anemia virus (EIAV) in the Brattleboro rat model of diabetes insipidus, in which a mutation in the arginine vasopressin (AVP) gene results in the production of nonfunctional mutant AVP precursor protein. Importantly, by using this model it is possible to monitor the success of the gene therapy treatment by noninvasive assays. Injection of an EIAV-CMV-AVP vector into the supraoptic nuclei of the hypothalamus resulted in expression of functional AVP peptide in magnocellular neurons. This was accompanied by a 100% recovery in water homeostasis as assessed by daily water intake, urine production, and urine osmolality lasting for a 1-year measurement period. These data show that a single gene defect leading to a neurological disorder can be corrected with a lentiviral-based strategy. This study highlights the potential of using viral gene therapy for the long-term treatment of disorders of the CNS.

  13. Contribution of VH Replacement Products in Mouse Antibody Repertoire

    PubMed Central

    Yu, Yangsheng; Li, Song; Su, Kaihong; Zhang, Zhixin

    2013-01-01

    VH replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS) near the 3′ end of a rearranged VH gene and the 23-bp RSS from an upstream unrearranged VH gene. Due to the location of the cRSS, VH replacement leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction, which can be used as a marker to identify VH replacement products. To determine the contribution of VH replacement products to mouse antibody repertoire, we developed a Java-based VH Replacement Footprint Analyzer (VHRFA) program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify VH replacement products. The overall frequency of VH replacement products in these IgH genes is 5.29% based on the identification of pentameric VH replacement footprints at their V-D junctions. The identified VH replacement products are distributed similarly in IgH genes using most families of VH genes, although different families of VH genes are used differentially. The frequencies of VH replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified VH replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice. PMID:23469094

  14. Phospholipase C gamma 1 (PLCG1) R707Q mutation is counterselected under targeted therapy in a patient with hepatic angiosarcoma

    PubMed Central

    Prenen, Hans; Smeets, Dominiek; Mazzone, Massimiliano; Lambrechts, Diether; Sagaert, Xavier; Sciot, Raf; Debiec-Rychter, Maria

    2015-01-01

    Hepatic angiosarcoma is a rare and aggressive vascular neoplasm. Pathogenic driver mutations are largely unknown. We present the case of a patient with recurrent hepatic angiosarcoma, who initially showed good response to sunitinib, followed by progression. Using comprehensive molecular techniques, we explored the potential mechanisms of resistance. By low-read-depth whole-genome sequencing, the comparison of copy number aberrations (CNAs) of the primary tumor to the skin metastatic lesion that developed after progression on sunitinib, revealed high-level amplification of the 4q11-q13.1 region (containing KIT, PDGFRA and VEGFR2 genes) that was sustained in both lesions. Whole exome sequencing on the germline, primary and metastatic tumor DNAs, resulted in 27 confirmed mutations, 19 of which (including TP53 mutation) presented in both primary and metastatic lesions. One mutation, ZNF331 frameshift deletion, was detected only in the primary tumor. In contrast, seven other mutations, including phospholipase C-gamma1 (PLCG1) R707Q mutation, were found only in the metastatic tumor, indicating selection of cells with the resistant genotype under sunitinib pressure. Our study supports the notion that PLCG1-R707Q mutation may confer VEGFR2-independent signaling and may thus cause resistance against VEGF(R)-directed therapies. This case illustrates also the advantages of using next-generation technologies in identifying individualized targeted therapy. PMID:26474454

  15. In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology.

    PubMed

    Fazli, Mustafa; Harrison, Joe J; Gambino, Michela; Givskov, Michael; Tolker-Nielsen, Tim

    2015-06-01

    Burkholderia cenocepacia is an emerging opportunistic pathogen causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis, which are often difficult, if not impossible, to treat. Understanding the genetic basis of virulence in this emerging pathogen is important for the development of novel treatment regimes. Generation of deletion mutations in genes predicted to encode virulence determinants is fundamental to investigating the mechanisms of pathogenesis. However, there is a lack of appropriate selectable and counterselectable markers for use in B. cenocepacia, making its genetic manipulation problematic. Here we describe a Gateway-compatible allelic exchange system based on the counterselectable pheS gene and the I-SceI homing endonuclease. This system provides efficiency in cloning homology regions of target genes and allows the generation of precise and unmarked gene deletions in B. cenocepacia. As a proof of concept, we demonstrate its utility by deleting the Bcam1349 gene, encoding a cyclic di-GMP (c-di-GMP)-responsive regulator protein important for biofilm formation.

  16. In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology

    PubMed Central

    Fazli, Mustafa; Harrison, Joe J.; Gambino, Michela; Givskov, Michael

    2015-01-01

    Burkholderia cenocepacia is an emerging opportunistic pathogen causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis, which are often difficult, if not impossible, to treat. Understanding the genetic basis of virulence in this emerging pathogen is important for the development of novel treatment regimes. Generation of deletion mutations in genes predicted to encode virulence determinants is fundamental to investigating the mechanisms of pathogenesis. However, there is a lack of appropriate selectable and counterselectable markers for use in B. cenocepacia, making its genetic manipulation problematic. Here we describe a Gateway-compatible allelic exchange system based on the counterselectable pheS gene and the I-SceI homing endonuclease. This system provides efficiency in cloning homology regions of target genes and allows the generation of precise and unmarked gene deletions in B. cenocepacia. As a proof of concept, we demonstrate its utility by deleting the Bcam1349 gene, encoding a cyclic di-GMP (c-di-GMP)-responsive regulator protein important for biofilm formation. PMID:25795676

  17. NifU and NifS are required for the maturation of nitrogenase and cannot replace the function of isc-gene products in Azotobacter vinelandii.

    PubMed

    Johnson, D C; Dos Santos, P C; Dean, D R

    2005-02-01

    In recent years, it has become evident that [Fe-S] proteins, such as hydrogenase, nitrogenase and aconitase, require a complex machinery to assemble and insert their associated [Fe-S] clusters. So far, three different types of [Fe-S] cluster biosynthetic systems have been identified and these have been designated nif, isc and suf. In the present work, we show that the nif-specific [Fe-S] cluster biosynthetic system from Azotobacter vinelandii, which is required for nitrogenase maturation, cannot functionally replace the isc [Fe-S] cluster system used for the maturation of other [Fe-S] proteins, such as aconitase. The results indicate that, in certain cases, [Fe-S] cluster biosynthetic machineries have evolved to perform only specialized functions.

  18. Impact of Fishmeal Replacement in Diets for Gilthead Sea Bream (Sparus aurata) on the Gastrointestinal Microbiota Determined by Pyrosequencing the 16S rRNA Gene

    PubMed Central

    Estruch, G.; Collado, M. C.; Peñaranda, D. S.; Tomás Vidal, A.; Jover Cerdá, M.; Pérez Martínez, G.; Martinez-Llorens, S.

    2015-01-01

    Recent studies have demonstrated the impact of diet on microbiota composition, but the essential need for the optimization of production rates and costs forces farms and aquaculture production to carry out continuous dietary tests. In order to understand the effect of total fishmeal replacement by vegetable-based feed in the sea bream (Sparus aurata), the microbial composition of the stomach, foregut, midgut and hindgut was analysed using high-throughput 16S rDNA sequencing, also considering parameters of growth, survival and nutrient utilisation indices.A total of 91,539 16S rRNA filtered-sequences were analysed, with an average number of 3661.56 taxonomically assigned, high-quality sequences per sample. The dominant phyla throughout the whole gastrointestinal tract were Actinobacteria, Protebacteria and Firmicutes. A lower diversity in the stomach in comparison to the other intestinal sections was observed. The microbial composition of the Recirculating Aquaculture System was totally different to that of the sea bream gastrointestinal tract. Total fishmeal replacement had an important impact on microbial profiles but not on diversity. Streptococcus (p-value: 0.043) and Photobacterium (p-value: 0.025) were highly represented in fish fed with fishmeal and vegetable-meal diets, respectively. In the stomach samples with the vegetable diet, reads of chloroplasts and mitochondria from vegetable dietary ingredients were rather abundant. Principal Coordinate Analysis showed a clear differentiation between diets in the microbiota present in the gut, supporting the presence of specific bacterial consortia associated with the diet.Although differences in growth and nutritive parameters were not observed, a negative effect of the vegetable diet on the survival rate was determined. Further studies are required to shed more light on the relationship between the immune system and sea bream gastrointestinal tract microbiota and should consider the modulation of the microbiota to

  19. Testing the critical window of estradiol replacement on gene expression of vasopressin, oxytocin, and their receptors, in the hypothalamus of aging female rats

    PubMed Central

    Garcia, Alexandra N.; Depena, Christina K.; Yin, Weiling; Gore, Andrea C.

    2015-01-01

    The current study tested the “critical window” hypothesis of menopause that postulates that the timing and duration of hormone treatment determine their potential outcomes. Our focus was genes in the rat hypothalamus involved in social and affiliative behaviors that change with aging and/or estradiol (E2): Avp, Avpr1a, Oxt, Oxtr, and Esr2 in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). Rats were reproductively mature or aging adults, ovariectomized, given E2 or vehicle treatment of different durations, with or without a post-OVX delay. Our hypothesis was that age-related changes in gene expression are mitigated by E2 treatments. Contrary to this, PVN Oxtr increased with E2, and Avpr1a increased with age. In the SON, Avpr1a increased with age, Oxtr with age and timing, and Avp was by duration. Thus, chronological age and E2 have independent actions on gene expression, with the “critical window” hypothesis supported by the observed timing and duration effects. PMID:26454088

  20. Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome

    PubMed Central

    Chang, Qing; Wang, Jianjun; Li, Qi; Kim, Yeunjung; Zhou, Binfei; Wang, Yunfeng; Li, Huawei; Lin, Xi

    2015-01-01

    Mutations in the potassium channel subunit KCNQ1 cause the human severe congenital deafness Jervell and Lange-Nielsen (JLN) syndrome. We applied a gene therapy approach in a mouse model of JLN syndrome (Kcnq1−/− mice) to prevent the development of deafness in the adult stage. A modified adeno-associated virus construct carrying a Kcnq1 expression cassette was injected postnatally (P0–P2) into the endolymph, which resulted in Kcnq1 expression in most cochlear marginal cells where native Kcnq1 is exclusively expressed. We also found that extensive ectopic virally mediated Kcnq1 transgene expression did not affect normal cochlear functions. Examination of cochlear morphology showed that the collapse of the Reissner’s membrane and degeneration of hair cells (HCs) and cells in the spiral ganglia were corrected in Kcnq1−/− mice. Electrophysiological tests showed normal endocochlear potential in treated ears. In addition, auditory brainstem responses showed significant hearing preservation in the injected ears, ranging from 20 dB improvement to complete correction of the deafness phenotype. Our results demonstrate the first successful gene therapy treatment for gene defects specifically affecting the function of the stria vascularis, which is a major site affected by genetic mutations in inherited hearing loss. PMID:26084842

  1. Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome.

    PubMed

    Chang, Qing; Wang, Jianjun; Li, Qi; Kim, Yeunjung; Zhou, Binfei; Wang, Yunfeng; Li, Huawei; Lin, Xi

    2015-08-01

    Mutations in the potassium channel subunit KCNQ1 cause the human severe congenital deafness Jervell and Lange-Nielsen (JLN) syndrome. We applied a gene therapy approach in a mouse model of JLN syndrome (Kcnq1(-/-) mice) to prevent the development of deafness in the adult stage. A modified adeno-associated virus construct carrying a Kcnq1 expression cassette was injected postnatally (P0-P2) into the endolymph, which resulted in Kcnq1 expression in most cochlear marginal cells where native Kcnq1 is exclusively expressed. We also found that extensive ectopic virally mediated Kcnq1 transgene expression did not affect normal cochlear functions. Examination of cochlear morphology showed that the collapse of the Reissner's membrane and degeneration of hair cells (HCs) and cells in the spiral ganglia were corrected in Kcnq1(-/-) mice. Electrophysiological tests showed normal endocochlear potential in treated ears. In addition, auditory brainstem responses showed significant hearing preservation in the injected ears, ranging from 20 dB improvement to complete correction of the deafness phenotype. Our results demonstrate the first successful gene therapy treatment for gene defects specifically affecting the function of the stria vascularis, which is a major site affected by genetic mutations in inherited hearing loss.

  2. Replacement of the human cytomegalovirus promoter with fish enhancer and core elements to control the expression of the G gene of viral haemorrhagic septicemia virus (VHSV).

    PubMed

    Martinez-Lopez, A; Chinchilla, B; Encinas, P; Gomez-Casado, E; Estepa, A; Coll, J M

    2012-12-15

    This work explores some of the possibilities to replace human cytomegalovirus (CMV) core and/or enhancer promoter control elements to create new expression vectors for use with fish. The work is relevant to fish vaccination, since DNA vaccines use eukaryotic expression plasmids controlled by the human cytomegalovirus (CMV) promoter to be effective against novirhabdoviruses, such as viral haemorrhagic septicemia virus (VHSV), one of the most devastating fish viral European diseases. To reduce possible homologous recombination with fish genome, core and enhancer sequences from fish origin, such as trout interferon-inducible myxovirus protein (Mx), zebrafish retrovirus long terminal repeat (LTR) and carp β-actin (AE6), were combined with those of CMV to design alternative hybrid promoters. The substitution of CMV core and/or enhancer with the corresponding elements of Mx or the LTR core maintained a similar in vitro protein G expression level than that obtained by using the CMV promoter. Vectors using the dsRNA-inducible Mx enhancer followed either by the LTR or the AE6 cores showed the highest in vitro protein G expression levels. Furthermore, synthetic constructs using the Mx enhancer maintained their polyI:C induction capabilities despite the core used. Some of these hybrid promoters might contribute to the development of all-fish-vectors for DNA vaccines while others might be useful for more basic studies.

  3. Spread of a new Chlamydia trachomatis variant from men who have sex with men to the heterosexual population after replacement and recombination in ompA and pmpH genes.

    PubMed

    Rodríguez-Domínguez, M; González-Alba, J M; Puerta, T; Martínez-García, L; Menéndez, B; Cantón, R; Del Romero, J; Galán, J C

    2017-10-01

    Sexually transmitted infections are frequently related to outbreaks in high-risk populations due to the dense sexual networks. We wanted to determine the dissemination of a Chlamydia trachomatis variant characterized by the pmpH-recombinant gene between L and G genotypes, which was previously described in a high-risk population. A total of 449 samples were analysed in two periods ranging from 2009 to 2015 for detection of the pmpH-recombinant gene. For those samples yielding positive amplification, a sampling was selected for phylogenetic reconstructions based on sequencing of five chromosomal genes. Globally this variant was found in 113 of the 449 samples (25%). During the first years (2009-13), this variant was found almost exclusively in rectal samples (30/112 samples) of men who have sex with men and in only one non-rectal sample (1/63). In 2014, this variant was also found in urethral and pharyngeal samples (1/24 and 1/7, respectively). However, in 2015, an epidemiological change was observed as the proportion of this variant had increased in rectal samples (20/51; 39%) and non-rectal samples, including cervical samples (51/142; 36.4%). The molecular characterization revealed the replacement of the ompA gene belonging to subtype G in samples recovered from 2009 to 2013 by the ompA gene belonging to subtype J after 2013. Our data would support the evidence that subtype J could be a 'subtype bridge' between different sexual networks, as subtype J has been found in men who have sex with men and heterosexual populations in similar proportions. This work reveals the necessity of implementing molecular surveillance in extra-rectal samples to help us understand the gaps in transmission. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  4. Radiation Source Replacement Workshop

    SciTech Connect

    Griffin, Jeffrey W.; Moran, Traci L.; Bond, Leonard J.

    2010-12-01

    This report summarizes a Radiation Source Replacement Workshop in Houston Texas on October 27-28, 2010, which provided a forum for industry and researchers to exchange information and to discuss the issues relating to replacement of AmBe, and potentially other isotope sources used in well logging.

  5. MDCA Needle 1 Replacement

    NASA Image and Video Library

    2013-04-22

    ISS035-E-025557(22 April 2013) ---Multi-user Droplet Combustion Apparatus (MDCA) Hardware Replacement: Cassidy accessed the Combustion Integration Rack (CIR) Combustion Chamber and removed the MDCA Chamber Insert Assembly (CIA). He then replaced the MDCA Needle 1 due to a fuel line that was damaged during previous activities when the MDCA CIA was being removed from the Combustion Chamber.

  6. Persistent transactivation by meis1 replaces hox function in myeloid leukemogenesis models: evidence for co-occupancy of meis1-pbx and hox-pbx complexes on promoters of leukemia-associated genes.

    PubMed

    Wang, Gang G; Pasillas, Martina P; Kamps, Mark P

    2006-05-01

    Homeobox transcription factors Meis1 and Hoxa9 promote hematopoietic progenitor self-renewal and cooperate to cause acute myeloid leukemia (AML). While Hoxa9 alone blocks the differentiation of nonleukemogenic myeloid cell-committed progenitors, coexpression with Meis1 is required for the production of AML-initiating progenitors, which also transcribe a group of hematopoietic stem cell genes, including Cd34 and Flt3 (defined as Meis1-related leukemic signature genes). Here, we use dominant trans-activating (Vp16 fusion) or trans-repressing (engrailed fusion) forms of Meis1 to define its biochemical functions that contribute to leukemogenesis. Surprisingly, Vp16-Meis1 (but not engrailed-Meis1) functioned as an autonomous oncoprotein that mimicked combined activities of Meis1 plus Hoxa9, immortalizing early progenitors, inducing low-level expression of Meis1-related signature genes, and causing leukemia without coexpression of exogenous or endogenous Hox genes. Vp16-Meis1-mediated transformation required the Meis1 function of binding to Pbx and DNA but not its C-terminal domain (CTD). The absence of endogenous Hox gene expression in Vp16-Meis1-immortalized progenitors allowed us to investigate how Hox alters gene expression and cell biology in early hematopoietic progenitors. Strikingly, expression of Hoxa9 or Hoxa7 stimulated both leukemic aggressiveness and transcription of Meis1-related signature genes in Vp16-Meis1 progenitors. Interestingly, while the Hoxa9 N-terminal domain (NTD) is essential for cooperative transformation with wild-type Meis1, it was dispensable in Vp16-Meis1 progenitors. The fact that a dominant transactivation domain fused to Meis1 replaces the essential functions of both the Meis1 CTD and Hoxa9 NTD suggests that Meis-Pbx and Hox-Pbx (or Hox-Pbx-Meis) complexes co-occupy cellular promoters that drive leukemogenesis and that Meis1 CTD and Hox NTD cooperate in gene activation. Chromatin immunoprecipitation confirmed co-occupancy of Hoxa9 and

  7. In vivo transduction by intravenous injection of a lentiviral vector expressing human ADA into neonatal ADA gene knockout mice: a novel form of enzyme replacement therapy for ADA deficiency.

    PubMed

    Carbonaro, Denise A; Jin, Xiangyang; Petersen, Denise; Wang, Xingchao; Dorey, Fred; Kil, Ki Soo; Aldrich, Melissa; Blackburn, Michael R; Kellems, Rodney E; Kohn, Donald B

    2006-06-01

    Using a mouse model of adenosine deaminase-deficient severe combined immune deficiency syndrome (ADA-deficient SCID), we have developed a noninvasive method of gene transfer for the sustained systemic expression of human ADA as enzyme replacement therapy. The method of delivery is a human immunodeficiency virus 1-based lentiviral vector given systemically by intravenous injection on day 1 to 2 of life. In this article we characterize the biodistribution of the integrated vector, the expression levels of ADA enzyme activity in various tissues, as well as the efficacy of systemic ADA expression to correct the ADA-deficient phenotype in this mouse model. The long-term expression of enzymatically active ADA achieved by this method, primarily from transduction of liver and lung, restored immunologic function and significantly extended survival. These studies illustrate the potential for sustained in vivo production of enzymatically active ADA, as an alternative to therapy by frequent injection of exogenous ADA protein.

  8. Histone replacement marks the boundaries of cis-regulatory domains.

    PubMed

    Mito, Yoshiko; Henikoff, Jorja G; Henikoff, Steven

    2007-03-09

    Cellular memory is maintained at homeotic genes by cis-regulatory elements whose mechanism of action is unknown. We have examined chromatin at Drosophila homeotic gene clusters by measuring, at high resolution, levels of histone replacement and nucleosome occupancy. Homeotic gene clusters display conspicuous peaks of histone replacement at boundaries of cis-regulatory domains superimposed over broad regions of low replacement. Peaks of histone replacement closely correspond to nuclease-hypersensitive sites, binding sites for Polycomb and trithorax group proteins, and sites of nucleosome depletion. Our results suggest the existence of a continuous process that disrupts nucleosomes and maintains accessibility of cis-regulatory elements.

  9. Cytochrome P450 (CYP2C9*2,*3) & vitamin-K epoxide reductase complex (VKORC1 -1639Ggene polymorphisms & their effect on acenocoumarol dose in patients with mechanical heart valve replacement

    PubMed Central

    Kaur, Anupriya; Khan, Farah; Agrawal, Suraksha S.; Kapoor, Aditya; Agarwal, Surendra K.; Phadke, Shubha R.

    2013-01-01

    Background & objectives: Studies have demonstrated the effect of CYP2C9 (cytochrome P450) and VKORC1 (vitamin K epoxide reductase complex) gene polymorphisms on the dose of acenocoumarol. The data from India about these gene polymorphisms and their effects on acenocoumarol dose are scarce. The aim of this study was to determine the occurrence of CYP2C9*2,*3 and VKORC 1 -1639G>A gene polymorphisms and to study their effects on the dose of acenocoumarol required to maintain a target International Normalized Ratio (INR) in patients with mechanical heart valve replacement. Methods: Patients from the anticoagulation clinic of a tertiary care hospital in north India were studied. The anticoagulation profile, INR (International Normalized Ratio) values and administered acenocoumarol dose were obtained from the clinical records of patients. Determination of the CYP2C9*2,*3 and VKORC1 -1639G>A genotypes was done by PCR-RFLP (restriction fragment length polymorphism). Results: A total of 111 patients were studied. The genotype frequencies of CYP2C9 *1/*1,*1/*2,*1/*3 were as 0.883, 0.072, 0.036 and that of VKORC1 -1639G>A for GG, AG, and AA genotypes were 0.883, 0.090, and 0.027, respectively. The percentage of patients carrying any of the variant alleles of CYP2C9 and VKORC1 in heterozygous or homozygous form was 34% among those receiving a low dose of ≤20 mg/wk while it was 13.8 per cent in those receiving >20 mg/wk (P=0.014). A tendency of lower dose requirements was seen among carriers of the studied polymorphisms. There was considerable variability in the dose requirements of patients with and without variant alleles. Interpretation & conclusions: The study findings point towards the role of CYP2C9 and VKORC1 gene polymorphisms in determining the inter-individual dose variability of acenocoumarol in the Indian patients with mechanical heart valve replacement. PMID:23481074

  10. A novel histone fold domain-containing protein that replaces TAF6 in Drosophila SAGA is required for SAGA-dependent gene expression

    PubMed Central

    Weake, Vikki M.; Swanson, Selene K.; Mushegian, Arcady; Florens, Laurence; Washburn, Michael P.; Abmayr, Susan M.; Workman, Jerry L.

    2009-01-01

    The histone acetyltransferase complex SAGA is well characterized as a coactivator complex in yeast. In this study of Drosophila SAGA (dSAGA), we describe three novel components that include an ortholog of Spt20, a potential ortholog of Sgf73/ATXN7, and a novel histone fold protein, SAF6 (SAGA factor-like TAF6). SAF6, which binds directly to TAF9, functions analogously in dSAGA to TAF6/TAF6L in the yeast and human SAGA complexes, respectively. Moreover, TAF6 in flies is restricted to TFIID. Mutations in saf6 disrupt SAGA-regulated gene expression without disrupting acetylated or ubiquitinated histone levels. Thus, SAF6 is essential for SAGA coactivator function independent of the enzymatic activities of the complex. PMID:20008933

  11. Two single nucleotide polymorphisms in the CYP17 and COMT Genes--relation to bone mass and longitudinal bone changes in postmenopausal women with or without hormone replacement therapy. The Danish Osteoporosis Prevention Study.

    PubMed

    Tofteng, C L; Abrahamsen, B; Jensen, J E B; Petersen, S; Teilmann, J; Kindmark, A; Vestergaard, P; Gram, J; Langdahl, B L; Mosekilde, L

    2004-08-01

    Sex steroids are important physiologic regulators of bone mass, and genes regulating sex steroid production and metabolism are obvious as candidate genes for osteoporosis susceptibility. We present data from a study of 1795 recent postmenopausal women, assigned to either hormone replacement therapy (HRT) or no treatment and followed for 5 years. The association between bone mass measurements and two single nucleotide polymorphisms, a T (A1) to C (A2) transition in the 5'-UTR of the cytochrome P450c17alpha (CYP17) gene and a G (Val) to A (Met) transition in exon 4 of the catechol- O-methyltransferase (COMT) gene, was evaluated. Association with CYP17 genotype was modified by body mass index (BMI). In lean women, individuals homozygous for the CYP17 A2 allele were 1 cm shorter and had lower baseline BMD (bone mineral density), BMC, and CSA (cross sectional area) in the spine and femoral neck than did other women (BMD spine A2A2: 0.975 g/cm2 versus 1.011 g/cm2 in A1A1 + A1A2, P = 0.002). Conversely, an adverse association with A2A2 and bone loss over 5 years seemed present only in overweight women, but differences were small. Response to HRT was not dependent on CYP17 genotype. COMT genotype was not associated with bone mass at baseline, bone loss in untreated women, or response to HRT. In conclusion, the A2 allele of the CYP17 T(27)-C polymorphism is associated with reduced bone mass and bone size in lean perimenopausal women, whereas high BMI protects against this negative association. The COMT G(1947)-A polymorphism is not associated with bone parameters in this study.

  12. Renal angiomyolipoma bleeding in a patient with TSC2/PKD1 contiguous gene syndrome after 17 years of renal replacement therapy.

    PubMed

    Furlano, Mónica; Barreiro, Yaima; Martí, Teresa; Facundo, Carme; Ruiz-García, César; DaSilva, Iara; Ayasreh, Nadia; Cabrera-López, Cristina; Ballarín, José; Ars, Elisabet; Torra, Roser

    We report the case of a 32-year-old male diagnosed with TSC2/PKD1 contiguous gene syndrome, presenting with tuberous sclerosis (TS) and autosomal dominant polycystic kidney disease simultaneously. He progressed to end-stage renal disease and received a kidney transplant at the age of 12. The native kidneys presented angiomyolipomas (AML), which are common benign tumours in patients with TS. Seventeen years after transplantation, he presented with abdominal pain, anaemia and a retroperitoneal haematoma, the latter caused by renal AML bleeding. Selective embolisation was performed. Our patient could have benefited from the administration of mTOR inhibitors at transplant. This therapy is immunosuppressive and reduces the size of benign tumours in TS as well as the risk of rupture and bleeding. This patient did not receive mTOR inhibitors at the time of the transplant because the relationship between mTOR inhibitors and TS was unknown at that time. This case confirms the persistent risk of renal AML bleeding for both transplanted patients and patients on dialysis. As a result, we would recommend routine check-ups of native kidneys and nephrectomy assessment. Copyright © 2017 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.

  13. Ankle replacement - discharge

    MedlinePlus

    ... ankle replacement. Your surgeon removed and reshaped damaged bones, and put in an artificial ankle joint. You received pain medicine and were shown how to treat swelling around your new ankle joint.

  14. Carbohydrates as Fat Replacers.

    PubMed

    Peng, Xingyun; Yao, Yuan

    2017-02-28

    The overconsumption of dietary fat contributes to various chronic diseases, which encourages attempts to develop and consume low-fat foods. Simple fat reduction causes quality losses that impede the acceptance of foods. Fat replacers are utilized to minimize the quality deterioration after fat reduction or removal to achieve low-calorie, low-fat claims. In this review, the forms of fats and their functions in contributing to food textural and sensory qualities are discussed in various food systems. The connections between fat reduction and quality loss are described in order to clarify the rationales of fat replacement. Carbohydrate fat replacers usually have low calorie density and provide gelling, thickening, stabilizing, and other texture-modifying properties. In this review, carbohydrates, including starches, maltodextrins, polydextrose, gums, and fibers, are discussed with regard to their interactions with other components in foods as well as their performances as fat replacers in various systems.

  15. Knee joint replacement - slideshow

    MedlinePlus

    ... presentations/100088.htm Knee joint replacement - series—Normal anatomy To use the sharing features on this page, ... Bethesda, MD 20894 U.S. Department of Health and Human Services National Institutes of Health Page last updated: ...

  16. Replacing a Missing Tooth

    MedlinePlus

    ... vessels in the tooth pulps are rather large. Drilling down these teeth for crowns may expose the ... porcelain replacement tooth is held in place by metal extensions cemented to the backs of the adjacent ...

  17. Artificial Disc Replacement

    MedlinePlus

    ... treat this condition, alternatives to disc replacement include fusion, nonoperative care or no treatment. Typically, surgery is ... operative treatment for disc pain has been spinal fusion. This is a surgical procedure in which disc ...

  18. Hormone Replacement Therapy

    MedlinePlus

    ... before and during menopause, the levels of female hormones can go up and down. This can cause ... hot flashes and vaginal dryness. Some women take hormone replacement therapy (HRT), also called menopausal hormone therapy, ...

  19. Artificial Disc Replacement

    MedlinePlus

    ... also disc replacements designed for use in the cervical spine (the neck). These devices have only been used ... of your spine increases the risk of significant injury during this type of spinal surgery. Back pain ...

  20. Nonlinear Pixel Replacement Estimation.

    DTIC Science & Technology

    1986-04-01

    Systems Command _________ _______________________ _____ 6e ADDRESS IC,,, Sra,. and ZIP Co~ de , 10 SOURCE Of FUNDING NUMBERS% RORMELEMENT NO PROJECT NO... de - scribed, this method does not replace array elements with computed values, but rather replaces them with one of the nine original pixel values. The...8217, gmx . NOISE PStd dev=’. sd call exit end include ’mathfunc For/nolist’ C-9 c biweighted 3xZ3 filter compute weighted mean from input iara subroutine

  1. Androgen replacement for women.

    PubMed Central

    Basson, R.

    1999-01-01

    OBJECTIVES: To determine whether a postmenopausal syndrome comprising specific changes in sexual desire and response associated with low free testosterone exists. To determine whether this syndrome is ameliorated by testosterone replacement. QUALITY OF EVIDENCE: Literature documenting that replacement of physiological levels of testosterone is beneficial and safe is scant. Only one randomized prospective blinded study examines sexual outcome in detail. MAIN MESSAGE: Testosterone is an important metabolic and sex hormone produced by the ovary throughout life. The variable reduction in ovarian testosterone production coincident with menopause is sometimes associated with a syndrome of specific changes in sexual desire and sexual response. Estrogen deficiency also impairs sexual response, but its replacement will not improve and might exacerbate sexual symptoms from androgen loss. Diagnosis of androgen deficiency is clinical, based on accurate assessment of a woman's sexual status before and after menopause and only confirmed (rather than diagnosed) by a low level of free testosterone. Partial androgen replacement restores much of the sexual response and facilitates sexual desire that is triggered by external cues. Avoiding supraphysiological levels of testosterone lessens risk of masculinization. Avoiding alkylated testosterone lessens hepatic or lipid impairment. CONCLUSION: Further prospective randomized studies of replacement of physiological levels of testosterone in women with androgen deficiency syndrome are needed, using formulations of testosterone available in Canada. The consistency of sexual changes, the associated personal and relationship distress, together with our clinical experience of the gratifying response to physiological replacement, make further studies urgently needed. PMID:10509222

  2. [Heterologous genes expression on Escherichia coli chromosome lac operon using Red recombination].

    PubMed

    Li, Shanhu; Shi, Qingguo; Huang, Cuifen; Zhou, Jianguang

    2008-04-01

    To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.

  3. RecET direct cloning and Redαβ recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression.

    PubMed

    Wang, Hailong; Li, Zhen; Jia, Ruonan; Hou, Yu; Yin, Jia; Bian, Xiaoying; Li, Aiying; Müller, Rolf; Stewart, A Francis; Fu, Jun; Zhang, Youming

    2016-07-01

    Full-length RecE and RecT from Rac prophage mediate highly efficient linear-linear homologous recombination that can be used to clone large DNA regions directly from genomic DNA into expression vectors, bypassing library construction and screening. Homologous recombination mediated by Redαβ from lambda phage has been widely used for recombinant DNA engineering. Here we present a protocol for direct cloning and engineering of biosynthetic gene clusters, large operons or single genes from genomic DNA using one Escherichia coli host that harbors both RecET and Redαβ systems. The pipeline uses standardized cassettes for horizontal gene transfer options, as well as vectors with different replication origins configured to minimize recombineering background through the use of selectively replicating templates or CcdB counterselection. These optimized reagents and protocols facilitate fast acquisition of transgenes from genomic DNA preparations, which are ready for heterologous expression within 1 week.

  4. Neural stem cells may be uniquely suited for combined gene therapy and cell replacement: Evidence from engraftment of Neurotrophin-3-expressing stem cells in hypoxic-ischemic brain injury.

    PubMed

    Park, Kook In; Himes, B Timothy; Stieg, Philip E; Tessler, Alan; Fischer, Itzhak; Snyder, Evan Y

    2006-05-01

    Previously, we reported that, when clonal neural stem cells (NSCs) were transplanted into brains of postnatal mice subjected to unilateral hypoxic-ischemic (HI) injury (optimally 3-7 days following infarction), donor-derived cells homed preferentially (from even distant locations) to and integrated extensively within the large ischemic areas that spanned the hemisphere. A subpopulation of NSCs and host cells, particularly in the penumbra, "shifted" their differentiation towards neurons and oligodendrocytes, the cell types typically damaged following asphyxia and least likely to regenerate spontaneously and in sufficient quantity in the "post-developmental" CNS. That no neurons and few oligodendrocytes were generated from the NSCs in intact postnatal cortex suggested that novel signals are transiently elaborated following HI to which NSCs might respond. The proportion of "replacement" neurons was approximately 5%. Neurotrophin-3 (NT-3) is known to play a role in inducing neuronal differentiation during development and perhaps following injury. We demonstrated that NSCs express functional TrkC receptors. Furthermore, the donor cells continued to express a foreign reporter transgene robustly within the damaged brain. Therefore, it appeared feasible that neuronal differentiation of exogenous NSCs (as well as endogenous progenitors) might be enhanced if donor NSCs were engineered prior to transplantation to (over)express a bioactive gene such as NT-3. A subclone of NSCs transduced with a retrovirus encoding NT-3 (yielding >90% neurons in vitro) was implanted into unilaterally asphyxiated postnatal day 7 mouse brain (emulating one of the common causes of cerebral palsy). The subclone expressed NT-3 efficiently in vivo. The proportion of NSC-derived neurons increased to approximately 20% in the infarction cavity and >80% in the penumbra. The neurons variously differentiated further into cholinergic, GABAergic, or glutamatergic subtypes, appropriate to the cortex. Donor

  5. Navigated unicompartmental knee replacement.

    PubMed

    Jenny, Jean-Yves

    2008-06-01

    Computer-aided systems have been developed recently to improve the precision of implantation of unicompartmental knee replacement (UKR) or total knee replacement. Minimal invasive techniques have been developed to decrease the surgical trauma related to the prosthesis implantation. However, there might be a concern about the potential of minimal invasive techniques for a loss of accuracy. Navigation systems might address this issue. We are currently using routinely a nonimage-based navigation for total knee replacement. We developed a modified system for UKR, suitable for either a conventional or a mini-invasive approach. Navigated implantation of a UKR allowed improving the accuracy of the radiologic implantation. Mini-invasive implantation was effective, but the accuracy may not reach that of the conventional navigated technique and should be still improved. Minimal invasive techniques have to be validated because a loss of accuracy will negatively influence long-term outcomes.

  6. Toxicology of chlorofluorocarbon replacements.

    PubMed Central

    Dekant, W

    1996-01-01

    Chlorofluorocarbons (CFCs) are stable in the atmosphere and may reach the stratosphere. They are cleaved by UV-radiation in the stratosphere to yield chlorine radicals, which are thought to interfere with the catalytic cycle of ozone formation and destruction and deplete stratospheric ozone concentrations. Due to potential adverse health effects of ozone depletion, chlorofluorocarbon replacements with much lower or absent ozone depleting potential are developed. The toxicology of these compounds that represent chlorofluorohydrocarbons (HCFCs) or fluorohydrocarbons (HFCs) has been intensively studied. All compounds investigated (1, 1-dichloro-1-fluoroethane [HCFC-141b], 1,1,1,2-tetrafluoroethane [HFC-134a], pentafluoroethane [HFC-125], 1-chloro- 1,2,2,2-tetrafluoroethane [HCFC-124], and 1,1-dichloro-2,2,2-trifluoroethane [HCFC-123]) show only a low potential for skin and eye irritation. Chronic adverse effects on the liver (HCFC-123) and the testes (HCFC-141b and HCFC-134a), including tumor formation, were observed in long-term inhalation studies in rodents using very high concentrations of these CFC replacements. All CFC replacements are, to varying extents, biotransformed in the organism, mainly by cytochrome P450-catalyzed oxidation of C-H bonds. The formed acyl halides are hydrolyzed to give excretable carboxylic acids; halogenated aldehydes that are formed may be further oxidized to halogenated carboxylic acids or reduced to halogenated alcohols, which are excretory metabolites in urine from rodents exposed experimentally to CFC replacements. The chronic toxicity of the CFC replacements studied is unlikely to be of relevance for humans exposed during production and application of CFC replacements. PMID:8722112

  7. Toxicology of chlorofluorocarbon replacements.

    PubMed

    Dekant, W

    1996-03-01

    Chlorofluorocarbons (CFCs) are stable in the atmosphere and may reach the stratosphere. They are cleaved by UV-radiation in the stratosphere to yield chlorine radicals, which are thought to interfere with the catalytic cycle of ozone formation and destruction and deplete stratospheric ozone concentrations. Due to potential adverse health effects of ozone depletion, chlorofluorocarbon replacements with much lower or absent ozone depleting potential are developed. The toxicology of these compounds that represent chlorofluorohydrocarbons (HCFCs) or fluorohydrocarbons (HFCs) has been intensively studied. All compounds investigated (1, 1-dichloro-1-fluoroethane [HCFC-141b], 1,1,1,2-tetrafluoroethane [HFC-134a], pentafluoroethane [HFC-125], 1-chloro- 1,2,2,2-tetrafluoroethane [HCFC-124], and 1,1-dichloro-2,2,2-trifluoroethane [HCFC-123]) show only a low potential for skin and eye irritation. Chronic adverse effects on the liver (HCFC-123) and the testes (HCFC-141b and HCFC-134a), including tumor formation, were observed in long-term inhalation studies in rodents using very high concentrations of these CFC replacements. All CFC replacements are, to varying extents, biotransformed in the organism, mainly by cytochrome P450-catalyzed oxidation of C-H bonds. The formed acyl halides are hydrolyzed to give excretable carboxylic acids; halogenated aldehydes that are formed may be further oxidized to halogenated carboxylic acids or reduced to halogenated alcohols, which are excretory metabolites in urine from rodents exposed experimentally to CFC replacements. The chronic toxicity of the CFC replacements studied is unlikely to be of relevance for humans exposed during production and application of CFC replacements.

  8. Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains.

    PubMed

    Bosma, Elleke F; van de Weijer, Antonius H P; van der Vlist, Laurens; de Vos, Willem M; van der Oost, John; van Kranenburg, Richard

    2015-07-07

    Microbial conversion of biomass to fuels or chemicals is an attractive alternative for fossil-based fuels and chemicals. Thermophilic microorganisms have several operational advantages as a production host over mesophilic organisms, such as low cooling costs, reduced contamination risks and a process temperature matching that of commercial hydrolytic enzymes, enabling simultaneous saccharification and fermentation at higher efficiencies and with less enzymes. However, genetic tools for biotechnologically relevant thermophiles are still in their infancy. In this study we developed a markerless gene deletion method for the thermophile Bacillus smithii and we report the first metabolic engineering of this species as a potential platform organism. Clean deletions of the ldhL gene were made in two B. smithii strains (DSM 4216(T) and compost isolate ET 138) by homologous recombination. Whereas both wild-type strains produced mainly L-lactate, deletion of the ldhL gene blocked L-lactate production and caused impaired anaerobic growth and acid production. To facilitate the mutagenesis process, we established a counter-selection system for efficient plasmid removal based on lacZ-mediated X-gal toxicity. This counter-selection system was applied to construct a sporulation-deficient B. smithii ΔldhL ΔsigF mutant strain. Next, we demonstrated that the system can be used repetitively by creating B. smithii triple mutant strain ET 138 ΔldhL ΔsigF ΔpdhA, from which also the gene encoding the α-subunit of the E1 component of the pyruvate dehydrogenase complex is deleted. This triple mutant strain produced no acetate and is auxotrophic for acetate, indicating that pyruvate dehydrogenase is the major route from pyruvate to acetyl-CoA. In this study, we developed a markerless gene deletion method including a counter-selection system for thermophilic B. smithii, constituting the first report of metabolic engineering in this species. The described markerless gene deletion system

  9. Biological tooth replacement

    PubMed Central

    Sartaj, Rachel; Sharpe, Paul

    2006-01-01

    Teeth develop from a series of reciprocal interactions that take place between epithelium and mesenchyme during development of the mouth that begin early in mammalian embryogenesis. The molecular control of key processes in tooth development such as initiation, morphogenesis and cytodifferentiation are being increasingly better understood, to the point where this information can be used as the basis for approaches to produce biological replacement teeth (BioTeeth). This review outlines the current approaches, ideas and progress towards the production of BioTeeth that could form an alternative method for replacing lost or damaged teeth. PMID:17005022

  10. Gene Therapy

    MedlinePlus

    ... cells in an effort to treat or stop disease. Genes contain your DNA — the code that controls much of your body's form and function, from making you grow taller to regulating your body systems. Genes that don't work properly can cause disease. Gene therapy replaces a faulty gene or adds ...

  11. Window Replacement Tips.

    ERIC Educational Resources Information Center

    Wiens, Janet

    2001-01-01

    Explores ways to replace facility windows more easily and efficiently by evaluating long-term needs and window options. Recommends obtaining references from window manufacturer and installer, scheduling the work during times the facility is unoccupied, and ensuring that historic and life-safety issues be considered. (GR)

  12. Prioritizing equipment for replacement.

    PubMed

    Capuano, Mike

    2010-01-01

    It is suggested that clinical engineers take the lead in formulating evaluation processes to recommend equipment replacement. Their skill, knowledge, and experience, combined with access to equipment databases, make them a logical choice. Based on ideas from Fennigkoh's scheme, elements such as age, vendor support, accumulated maintenance cost, and function/risk were used.6 Other more subjective criteria such as cost benefits and efficacy of newer technology were not used. The element of downtime was also omitted due to the data element not being available. The resulting Periop Master Equipment List and its rationale was presented to the Perioperative Services Program Council. They deemed the criteria to be robust and provided overwhelming acceptance of the list. It was quickly put to use to estimate required capital funding, justify items already thought to need replacement, and identify high-priority ranked items for replacement. Incorporating prioritization criteria into an existing equipment database would be ideal. Some commercially available systems do have the basic elements of this. Maintaining replacement data can be labor-intensive regardless of the method used. There is usually little time to perform the tasks necessary for prioritizing equipment. However, where appropriate, a clinical engineering department might be able to conduct such an exercise as shown in the following case study.

  13. Python import replacement

    SciTech Connect

    2011-10-01

    SmartImport.py is a Python source-code file that implements a replacement for the standard Python module importer. The code is derived from knee.py, a file in the standard Python diestribution , and adds functionality to improve the performance of Python module imports in massively parallel contexts.

  14. Hexachloroethane Obscurant Replacement

    DTIC Science & Technology

    2012-01-01

    suggest that neither the alkali metal hydroxides nor chlorides are extremely toxic (11). Oral rat LD50 values are indeed lower for hydroxide salts...analyze for toxicity of combustion gases and particulates. Compositions containing boron potassium nitrate igniter blended with halide salts...variants were identified which should have improved performance. 15. SUBJECT TERMS Non- toxic obscurant, hexachloroethane smoke replacement 16

  15. Python import replacement

    SciTech Connect

    2011-10-01

    SmartImport.py is a Python source-code file that implements a replacement for the standard Python module importer. The code is derived from knee.py, a file in the standard Python diestribution , and adds functionality to improve the performance of Python module imports in massively parallel contexts.

  16. Replacing America's Job Bank

    ERIC Educational Resources Information Center

    Vollman, Jim

    2009-01-01

    The Job Central National Labor Exchange (www.jobcentral.com) has become the effective replacement for America's Job Bank with state workforce agencies and, increasingly, with community colleges throughout the country. The American Association of Community Colleges (AACC) has formed a partnership with Job Central to promote its use throughout the…

  17. Replacing America's Job Bank

    ERIC Educational Resources Information Center

    Vollman, Jim

    2009-01-01

    The Job Central National Labor Exchange (www.jobcentral.com) has become the effective replacement for America's Job Bank with state workforce agencies and, increasingly, with community colleges throughout the country. The American Association of Community Colleges (AACC) has formed a partnership with Job Central to promote its use throughout the…

  18. ARRANGEMENT FOR REPLACING FILTERS

    DOEpatents

    Blomgren, R.A.; Bohlin, N.J.C.

    1957-08-27

    An improved filtered air exhaust system which may be continually operated during the replacement of the filters without the escape of unfiltered air is described. This is accomplished by hermetically sealing the box like filter containers in a rectangular tunnel with neoprene covered sponge rubber sealing rings coated with a silicone impregnated pneumatic grease. The tunnel through which the filters are pushed is normal to the exhaust air duct. A number of unused filters are in line behind the filters in use, and are moved by a hydraulic ram so that a fresh filter is positioned in the air duct. The used filter is pushed into a waiting receptacle and is suitably disposed. This device permits a rapid and safe replacement of a radiation contaminated filter without interruption to the normal flow of exhaust air.

  19. Celotex (Registered) Replacement Study

    SciTech Connect

    Couture, S; Hafner, R

    2002-10-01

    The AL-R8 is the pit storage container in most widespread use at Pantex. The AL-R8 container family consists of standard 20-in.-diameter steel drums, 30 to 60 in. in height, with insulation inserts made of Celotex{reg_sign}--a fiberboard product made from processed sugar cane. Celotex is an acceptable material for inserts in many radioactive material shipping and storage containers. It is a good shock mitigator/insulator, does a fair job in fire protection (when oxygen is excluded), shielding, and criticality, and is inexpensive and easily available. However, the fiberboard absorbs water in humid environments which, when combined with chemical residues in the fiberboard, forms corrosive compounds that can shorten the life of the container and affect container contents. To protect the contents from this potentially damaging environment, the AL-R8 SI was developed to isolate the contents within a sealed stainless steel vessel inside the AL-R8. Although the SI protected the contents, corrosion studies indicated the SI lid bolts might corrode over time and surveillance showed that areas of the outer drum were still subject to corrosion. To address this potential problem, DOE/Albuquerque sponsored bolt and Celotex replacement studies. The bolt replacement study was assigned to Mason and Hanger's Pantex Facility and this Celotex Replacement Study to Lawrence Livermore National Laboratory. The Celotex Replacement Study evaluated options for replacing Celotex with a material that is chemically compatible with the AL-R8 SI container. The evaluation was limited to materials either used previously in nuclear materials shipping and storage containers or materials with known properties in a low-radiation environment. This limitation was set to ensure that the long-term aging effect on the new material is known a priori. Initial material evaluations narrowed the material choices to foam and cork. Although cork performed better than foam in some tests, cork was considered a less

  20. NARC Rayon Replacement Program

    NASA Technical Reports Server (NTRS)

    Wilson, Kenneth P.; McCool, Alex (Technical Monitor)

    2002-01-01

    This viewgraph presentation provides information on the search for a replacement product for the aerospace grade rayon formerly used in the reusable solid rocket motor (RSRM). The rayon used in RSRMs is no longer produced by the North American Rayon Corporation (NARC), though NASA has stockpiled rayon to be used until a suitable replacement is found to be used in the RSRM nozzle carbon cloth phenolic (CCP). The primary considerations are performance, process ability, variability, long term availability, and cost. The preference will be given to commercial off-the-shelf products as opposed to those which are custom made. The candidate materials have been tested in previous phases of this selection process, and Enka Textile Rayon has emerged as a possible fabric, though its availability is still in question.

  1. Ulnar head replacement.

    PubMed

    Herbert, Timothy J; van Schoonhoven, Joerg

    2007-03-01

    Recent years have seen an increasing awareness of the anatomical and biomechanical significance of the distal radioulnar joint (DRUJ). With this has come a more critical approach to surgical management of DRUJ disorders and a realization that all forms of "excision arthroplasty" can only restore forearm rotation at the expense of forearm stability. This, in turn, has led to renewed interest in prosthetic replacement of the ulnar head, a procedure that had previously fallen into disrepute because of material failures with early implants, in particular, the Swanson silicone ulnar head replacement. In response to these early failures, a new prosthesis was developed in the early 1990s, using materials designed to withstand the loads across the DRUJ associated with normal functional use of the upper limb. Released onto the market in 1995 (Herbert ulnar head prosthesis), clinical experience during the last 10 years has shown that this prosthesis is able to restore forearm function after ulnar head excision and that the materials (ceramic head and noncemented titanium stem), even with normal use of the limb, are showing no signs of failure in the medium to long term. As experience with the use of an ulnar head prosthesis grows, so does its acceptance as a viable and attractive alternative to more traditional operations, such as the Darrach and Sauve-Kapandji procedures. This article discusses the current indications and contraindications for ulnar head replacement and details the surgical procedure, rehabilitation, and likely outcomes.

  2. Renal Replacement Therapy

    PubMed Central

    Ricci, Zaccaria; Romagnoli, Stefano; Ronco, Claudio

    2016-01-01

    During the last few years, due to medical and surgical evolution, patients with increasingly severe diseases causing multiorgan dysfunction are frequently admitted to intensive care units. Therapeutic options, when organ failure occurs, are frequently nonspecific and mostly directed towards supporting vital function. In these scenarios, the kidneys are almost always involved and, therefore, renal replacement therapies have become a common routine practice in critically ill patients with acute kidney injury. Recent technological improvement has led to the production of safe, versatile and efficient dialysis machines. In addition, emerging evidence may allow better individualization of treatment with tailored prescription depending on the patients’ clinical picture (e.g. sepsis, fluid overload, pediatric). The aim of the present review is to give a general overview of current practice in renal replacement therapies for critically ill patients. The main clinical aspects, including dose prescription, modality of dialysis delivery, anticoagulation strategies and timing will be addressed. In addition, some technical issues on physical principles governing blood purification, filters characteristics, and vascular access, will be covered. Finally, a section on current standard nomenclature of renal replacement therapy is devoted to clarify the “Tower of Babel” of critical care nephrology. PMID:26918174

  3. Replacement steel windows

    SciTech Connect

    Brown, M.A.; Condren, S.J.

    1999-07-01

    This paper presents the authors experiences in the investigation, design, and installation of replacement steel windows for two renovation projects at a major university in the northeast: a student residential complex and the law school. The authors review the construction of the existing walls (cast-in-place concrete barrier wall at the student residential complex and brick and stone masonry barrier wall at the law school), and the construction and performance of the original steel windows. To maintain the appearance of these architecturally significant buildings, the university elected to install replacement steel windows. The authors discuss special design consideration for steel windows (versus the more prevalent aluminum replacement window), including available window section profiles, corrosion protection, frame fabrication, and glazing design. The authors also review window flashing concepts they employed for the barrier wall construction, which has no drainage cavity. The authors summarize lessons learned during the window selection, design, fabrication, testing, and installation phases of the projects, and present recommendations for improved durability and water penetration resistance of steel windows.

  4. Drosophila lines with mutant and wild type human TDP-43 replacing the endogenous gene reveals phosphorylation and ubiquitination in mutant lines in the absence of viability or lifespan defects

    PubMed Central

    Chang, Jer-Cherng

    2017-01-01

    Mutations in TDP-43 are associated with proteinaceous inclusions in neurons and are believed to be causative in neurodegenerative diseases such as frontotemporal dementia or amyotrophic lateral sclerosis. Here we describe a Drosophila system where we have engineered the genome to replace the endogenous TDP-43 orthologue with wild type or mutant human TDP-43(hTDP-43). In contrast to other models, these flies express both mutant and wild type hTDP-43 at similar levels to those of the endogenous gene and importantly, no age-related TDP-43 accumulation observed among all the transgenic fly lines. Immunoprecipitation of TDP-43 showed that flies with hTDP-43 mutations had increased levels of ubiquitination and phosphorylation of the hTDP-43 protein. Furthermore, histologically, flies expressing hTDP-43 M337V showed global, robust neuronal staining for phospho-TDP. All three lines: wild type hTDP-43, -G294A and -M337V were homozygous viable, with no defects in development, life span or behaviors observed. The primary behavioral defect was that flies expressing either hTDP-43 G294A or M337V showed a faster decline with age in negative geotaxis. Together, these observations implied that neurons could handle these TDP-43 mutations by phosphorylation- and ubiquitin-dependent proteasome systems, even in a background without the wild type TDP-43. Our findings suggest that these two specific TDP-43 mutations are not inherently toxic, but may require additional environmental or genetic factors to affect longevity or survival. PMID:28686708

  5. Drosophila lines with mutant and wild type human TDP-43 replacing the endogenous gene reveals phosphorylation and ubiquitination in mutant lines in the absence of viability or lifespan defects.

    PubMed

    Chang, Jer-Cherng; Morton, David B

    2017-01-01

    Mutations in TDP-43 are associated with proteinaceous inclusions in neurons and are believed to be causative in neurodegenerative diseases such as frontotemporal dementia or amyotrophic lateral sclerosis. Here we describe a Drosophila system where we have engineered the genome to replace the endogenous TDP-43 orthologue with wild type or mutant human TDP-43(hTDP-43). In contrast to other models, these flies express both mutant and wild type hTDP-43 at similar levels to those of the endogenous gene and importantly, no age-related TDP-43 accumulation observed among all the transgenic fly lines. Immunoprecipitation of TDP-43 showed that flies with hTDP-43 mutations had increased levels of ubiquitination and phosphorylation of the hTDP-43 protein. Furthermore, histologically, flies expressing hTDP-43 M337V showed global, robust neuronal staining for phospho-TDP. All three lines: wild type hTDP-43, -G294A and -M337V were homozygous viable, with no defects in development, life span or behaviors observed. The primary behavioral defect was that flies expressing either hTDP-43 G294A or M337V showed a faster decline with age in negative geotaxis. Together, these observations implied that neurons could handle these TDP-43 mutations by phosphorylation- and ubiquitin-dependent proteasome systems, even in a background without the wild type TDP-43. Our findings suggest that these two specific TDP-43 mutations are not inherently toxic, but may require additional environmental or genetic factors to affect longevity or survival.

  6. Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27

    PubMed Central

    Wang, Lei; Hoffmann, Jana; Watzlawick, Hildegard

    2015-01-01

    We developed a counterselectable deletion system for Thermus thermophilus HB27 based on cytosine deaminase (encoded by codA) from Thermaerobacter marianensis DSM 12885 and the sensitivity of T. thermophilus HB27 to the antimetabolite 5-fluorocytosine (5-FC). The deletion vector comprises the pUC18 origin of replication, a thermostable kanamycin resistance marker functional in T. thermophilus HB27, and codA under the control of a constitutive putative trehalose promoter from T. thermophilus HB27. The functionality of the system was demonstrated by deletion of the bglT gene, encoding a β-glycosidase, and three carotenoid biosynthesis genes, CYP175A1, crtY, and crtI, from the genome of T. thermophilus HB27. PMID:26655764

  7. Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27.

    PubMed

    Wang, Lei; Hoffmann, Jana; Watzlawick, Hildegard; Altenbuchner, Josef

    2015-12-11

    We developed a counterselectable deletion system for Thermus thermophilus HB27 based on cytosine deaminase (encoded by codA) from Thermaerobacter marianensis DSM 12885 and the sensitivity of T. thermophilus HB27 to the antimetabolite 5-fluorocytosine (5-FC). The deletion vector comprises the pUC18 origin of replication, a thermostable kanamycin resistance marker functional in T. thermophilus HB27, and codA under the control of a constitutive putative trehalose promoter from T. thermophilus HB27. The functionality of the system was demonstrated by deletion of the bglT gene, encoding a β-glycosidase, and three carotenoid biosynthesis genes, CYP175A1, crtY, and crtI, from the genome of T. thermophilus HB27. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  8. CoCr wear particles generated from CoCr alloy metal-on-metal hip replacements, and cobalt ions stimulate apoptosis and expression of general toxicology-related genes in monocyte-like U937 cells

    SciTech Connect

    Posada, Olga M.; Gilmour, Denise; Tate, Rothwelle J.; Grant, M. Helen

    2014-11-15

    Cobalt-chromium (CoCr) particles in the nanometre size range and their concomitant release of Co and Cr ions into the patients' circulation are produced by wear at the articulating surfaces of metal-on-metal (MoM) implants. This process is associated with inflammation, bone loss and implant loosening and led to the withdrawal from the market of the DePuy ASR™ MoM hip replacements in 2010. Ions released from CoCr particles derived from a resurfacing implant in vitro and their subsequent cellular up-take were measured by ICP-MS. Moreover, the ability of such metal debris and Co ions to induce both apoptosis was evaluated with both FACS and immunoblotting. qRT-PCR was used to assess the effects on the expression of lymphotoxin alpha (LTA), BCL2-associated athanogene (BAG1), nitric oxide synthase 2 inducible (NOS2), FBJ murine osteosarcoma viral oncogene homolog (FOS), growth arrest and DNA-damage-inducible alpha (GADD45A). ICP-MS showed that the wear debris released significant (p < 0.05) amounts of Co and Cr ions into the culture medium, and significant (p < 0.05) cellular uptake of both ions. There was also an increase (p < 0.05) in apoptosis after a 48 h exposure to wear debris. Analysis of qRT-PCR results found significant up-regulation (p < 0.05) particularly of NOS2 and BAG1 in Co pre-treated cells which were subsequently exposed to Co ions + debris. Metal debris was more effective as an inducer of apoptosis and gene expression when cells had been pre-treated with Co ions. This suggests that if a patient receives sequential bilateral CoCr implants, the second implant may be more likely to produce adverse effects than the first one. - Highlights: • Effects of CoCr nanoparticles and Co ions on U937 cells were investigated. • Ions released from wear debris play an important role in cellular response, • Toxicity of Co ions could be related to NO metabolic processes and apoptosis. • CoCr particles were a more effective inducer of apoptosis after cell

  9. Total ankle joint replacement.

    PubMed

    2016-02-01

    Ankle arthritis results in a stiff and painful ankle and can be a major cause of disability. For people with end-stage ankle arthritis, arthrodesis (ankle fusion) is effective at reducing pain in the shorter term, but results in a fixed joint, and over time the loss of mobility places stress on other joints in the foot that may lead to arthritis, pain and dysfunction. Another option is to perform a total ankle joint replacement, with the aim of giving the patient a mobile and pain-free ankle. In this article we review the efficacy of this procedure, including how it compares to ankle arthrodesis, and consider the indications and complications.

  10. Immediate total tooth replacement.

    PubMed

    Garber, D A; Salama, M A; Salama, H

    2001-03-01

    Successful implant placement at the time of extraction has been documented. Implant placement at the time of extraction was initially performed as a two-stage procedure often with barrier membranes and sophisticated second-stage surgical uncoverings. The authors describe the next generation of this technique, including atraumatic tooth removal with simultaneous root form, implant placement, and temporization at one appointment. This technique of "Immediate Total Tooth Replacement" allows for the maintenance of the bony housing and soft-tissue form that existed before extraction, while at the same time establishing a root form anchor in the bone for an esthetic restoration.

  11. [Hormone replacement therapy].

    PubMed

    Nozue, E

    1994-03-01

    Hormone replacement therapy after menopause has shown so many advantages, such as reduction in risk of coronary artery disease and osteoporotic fracture besides treatment for climacteric symptoms, yet disadvantage such as a probability of an increase in the risk of breast cancer with long-term use still remains. In Japan HRT is now at the starting point. We must try to make more effort to accumulate data on the safety of HRT during-and-after menopause. It is our hope that doctors, not only gynecologists, but also doctors of other specialties will join our program for the health and happiness of women after menopause.

  12. Search for genes essential for pneumococcal transformation: the RADA DNA repair protein plays a role in genomic recombination of donor DNA.

    PubMed

    Burghout, Peter; Bootsma, Hester J; Kloosterman, Tomas G; Bijlsma, Jetta J E; de Jongh, Christa E; Kuipers, Oscar P; Hermans, Peter W M

    2007-09-01

    We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counterselection of mutants in 21 different genes during the challenge. Eight of the counterselected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants of seven of the eight remaining genes, i.e., spr0459-spr0460, spr0777, spr0838, spr1259-spr1260, and spr1357, resulted in reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation with chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and in DNA damage repair in S. pneumoniae.

  13. Cadmium plating replacements

    NASA Technical Reports Server (NTRS)

    Nelson, Mary J.; Groshart, Earl C.

    1995-01-01

    The Boeing Company has been searching for replacements to cadmium plate. Two alloy plating systems seem close to meeting the needs of a cadmium replacement. The two alloys, zinc-nickel and tin-zinc are from alloy plating baths; both baths are neutral pH. The alloys meet the requirements for salt fog corrosion resistance, and both alloys excel as a paint base. Currently, tests are being performed on standard fasteners to compare zinc-nickel and tin-zinc on threaded hardware where cadmium is heavily used. The Hydrogen embrittlement propensity of the zinc-nickel bath has been tested, and just beginning for the tin-zinc bath. Another area of interest is the electrical properties on aluminum for tin-zinc and will be discussed. The zinc-nickel alloy plating bath is in production in Boeing Commercial Airplane Group for non-critical low strength steels. The outlook is promising that these two coatings will help The Boeing Company significantly reduce its dependence on cadmium plating.

  14. Sutureless aortic valve replacement

    PubMed Central

    Phan, Kevin

    2015-01-01

    The increasing incidence of aortic stenosis and greater co-morbidities and risk profiles of the contemporary patient population has driven the development of minimally invasive aortic valve surgery and percutaneous transcatheter aortic valve implantation (TAVI) techniques to reduce surgical trauma. Recent technological developments have led to an alternative minimally invasive option which avoids the placement and tying of sutures, known as “sutureless” or rapid deployment aortic valves. Potential advantages for sutureless aortic prostheses include reducing cross-clamp and cardiopulmonary bypass (CPB) duration, facilitating minimally invasive surgery and complex cardiac interventions, whilst maintaining satisfactory hemodynamic outcomes and low paravalvular leak rates. However, given its recent developments, the majority of evidence regarding sutureless aortic valve replacement (SU-AVR) is limited to observational studies and there is a paucity of adequately-powered randomized studies. Recently, the International Valvular Surgery Study Group (IVSSG) has formulated to conduct the Sutureless Projects, set to be the largest international collaborative group to investigate this technology. This keynote lecture will overview the use, the potential advantages, the caveats, and current evidence of sutureless and rapid deployment aortic valve replacement (AVR). PMID:25870807

  15. Power Plant Replacement Study

    SciTech Connect

    Reed, Gary

    2010-09-30

    This report represents the final report for the Eastern Illinois University power plant replacement study. It contains all related documentation from consideration of possible solutions to the final recommended option. Included are the economic justifications associated with the chosen solution along with application for environmental permitting for the selected project for construction. This final report will summarize the results of execution of an EPC (energy performance contract) investment grade audit (IGA) which lead to an energy services agreement (ESA). The project includes scope of work to design and install energy conservation measures which are guaranteed by the contractor to be self-funding over its twenty year contract duration. The cost recovery is derived from systems performance improvements leading to energy savings. The prime focus of this EPC effort is to provide a replacement solution for Eastern Illinois University's aging and failing circa 1925 central steam production plant. Twenty-three ECMs were considered viable whose net impact will provide sufficient savings to successfully support the overall project objectives.

  16. Power Plant Replacement Study

    SciTech Connect

    Reed, Gary

    2010-09-30

    This report represents the final report for the Eastern Illinois University power plant replacement study. It contains all related documentation from consideration of possible solutions to the final recommended option. Included are the economic justifications associated with the chosen solution along with application for environmental permitting for the selected project for construction. This final report will summarize the results of execution of an EPC (energy performance contract) investment grade audit (IGA) which lead to an energy services agreement (ESA). The project includes scope of work to design and install energy conservation measures which are guaranteed by the contractor to be self-funding over its twenty year contract duration. The cost recovery is derived from systems performance improvements leading to energy savings. The prime focus of this EPC effort is to provide a replacement solution for Eastern Illinois University’s aging and failing circa 1925 central steam production plant. Twenty-three ECMs were considered viable whose net impact will provide sufficient savings to successfully support the overall project objectives.

  17. Cadmium plating replacements

    SciTech Connect

    Nelson, M.J.; Groshart, E.C.

    1995-03-01

    The Boeing Company has been searching for replacements to cadmium plate. Two alloy plating systems seem close to meeting the needs of a cadmium replacement. The two alloys, zinc-nickel and tin-zinc are from alloy plating baths; both baths are neutral pH. The alloys meet the requirements for salt fog corrosion resistance, and both alloys excel as a paint base. Currently, tests are being performed on standard fasteners to compare zinc-nickel and tin-zinc on threaded hardware where cadmium is heavily used. The Hydrogen embrittlement propensity of the zinc-nickel bath has been tested, and just beginning for the tin-zinc bath. Another area of interest is the electrical properties on aluminum for tin-zinc and will be discussed. The zinc-nickel alloy plating bath is in production in Boeing Commercial Airplane Group for non-critical low strength steels. The outlook is promising that these two coatings will help The Boeing Company significantly reduce its dependence on cadmium plating.

  18. Power Plant Replacement Study

    SciTech Connect

    Reed, Gary

    2010-09-30

    This report represents the final report for the Eastern Illinois University power plant replacement study. It contains all related documentation from consideration of possible solutions to the final recommended option. Included are the economic justifications associated with the chosen solution along with application for environmental permitting for the selected project for construction. This final report will summarize the results of execution of an EPC (energy performance contract) investment grade audit (IGA) which lead to an energy services agreement (ESA). The project includes scope of work to design and install energy conservation measures which are guaranteed by the contractor to be self-funding over its twenty year contract duration. The cost recovery is derived from systems performance improvements leading to energy savings. The prime focus of this EPC effort is to provide a replacement solution for Eastern Illinois University’s aging and failing circa 1925 central steam production plant. Twenty-three ECMs were considered viable whose net impact will provide sufficient savings to successfully support the overall project objectives.

  19. Power Plant Replacement Study

    SciTech Connect

    Reed, Gary

    2010-09-30

    This report represents the final report for the Eastern Illinois University power plant replacement study. It contains all related documentation from consideration of possible solutions to the final recommended option. Included are the economic justifications associated with the chosen solution along with application for environmental permitting for the selected project for construction. This final report will summarize the results of execution of an EPC (energy performance contract) investment grade audit (IGA) which lead to an energy services agreement (ESA). The project includes scope of work to design and install energy conservation measures which are guaranteed by the contractor to be self‐funding over its twenty year contract duration. The cost recovery is derived from systems performance improvements leading to energy savings. The prime focus of this EPC effort is to provide a replacement solution for Eastern Illinois University’s aging and failing circa 1925 central steam production plant. Twenty‐three ECMs were considered viable whose net impact will provide sufficient savings to successfully support the overall project objectives.

  20. Total disc replacement.

    PubMed

    Vital, J-M; Boissière, L

    2014-02-01

    Total disc replacement (TDR) (partial disc replacement will not be described) has been used in the lumbar spine since the 1980s, and more recently in the cervical spine. Although the biomechanical concepts are the same and both are inserted through an anterior approach, lumbar TDR is conventionally indicated for chronic low back pain, whereas cervical TDR is used for soft discal hernia resulting in cervicobrachial neuralgia. The insertion technique must be rigorous, with precise centering in the disc space, taking account of vascular anatomy, which is more complex in the lumbar region, particularly proximally to L5-S1. All of the numerous studies, including prospective randomized comparative trials, have demonstrated non-inferiority to fusion, or even short-term superiority regarding speed of improvement. The main implant-related complication is bridging heterotopic ossification with resulting loss of range of motion and increased rates of adjacent segment degeneration, although with an incidence lower than after arthrodesis. A sufficiently long follow-up, which has not yet been reached, will be necessary to establish definitively an advantage for TDR, particularly in the cervical spine. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  1. Hormone replacement therapy and longevity.

    PubMed

    Comhaire, F

    2016-02-01

    To assess whether hormone replacement therapy influences longevity, an analysis was made of published life tables allowing for the calculation of the relative benefit of hormone replacement therapy on longevity in men with late onset hypogonadism and in post-menopausal women. It was found that testosterone replacement therapy of men suffering from late onset hypogonadism increased survival rate by 9-10% in 5 years, similar to that of eugonadal, non-LOH men with normal endogenous testosterone secretion. Oestrogen replacement therapy resulted in increased survival by 2.6% in 5 years. It is concluded that hormone replacement therapy increases longevity.

  2. Functional replacements for gluten.

    PubMed

    Zannini, Emanuele; Jones, Julie Miller; Renzetti, Stefano; Arendt, Elke K

    2012-01-01

    Celiac disease (CD) is an immune-mediated disease triggered in genetically susceptible individuals by ingested gluten from wheat, rye, barley, and other closely related cereal grains. Currently, the only therapy able to normalize the clinical and histological manifestation of the disease is a strict and life-long gluten-free (GF) diet. The replacement of gluten presents a significant technological challenge, as it is an essential structure-building protein, which is necessary for formulating high-quality baked goods. The objective of this paper is to review some basics about CD, its current prevalence, and the recent advances in the preparation of high-quality GF breads using GF flours, starches, hydrocolloids, gums, and novel functional ingredients and technologies.

  3. Antenna grout replacement system

    NASA Technical Reports Server (NTRS)

    Mcclung, C. E. (Inventor)

    1983-01-01

    An epoxy grout suitable for use in mounting and positioning bearing runner plates used in hydrostatic bearing assemblies for rotatably mounting large radio telescope structures to stationary support pedestals is described. The epoxy grout may be used in original mountings or may be used as part of a replacement system for repairing cavities in existing grout resulting from grout deterioration. The epoxy grout has a relatively short work life and cure time even in the presence of hydraulic oil. The epoxy grout cures without shrinking or sagging to form a grout which is sufficiently strong and durable to provide a grout especially well suited for use under the high pressure loading and close tolerance requirements of large hydrostatic bearing assemblies.

  4. Aerocoat 7 Replacement Coatings

    NASA Technical Reports Server (NTRS)

    2008-01-01

    Kennedy Space Center has used Aerocoat 7 (AR-7) to protect stainless-steel flex hoses at Launch Complex (LC-39) and hydraulic lines of the Mobile Launcher Platform (MLP) because it provides excellent corrosion protection in low-temperature applications. The Sovereign Company produced AR-7 exclusively for NASA but discontinued production because the coating released high levels of volatile organic compounds (VOCs) and had a significant environmental impact. The purpose of this project was to select and evaluate potential replacement coatings for AR-7 that would be more environmentally sound. The physical and mechanical properties of commercially available coatings were investigated through the Internet. The ideal coating would be fluid enough to penetrate the outer mesh of a stainless-steel flex hose and coat the inner hose, and flexible enough to withstand the movement of the hose, as well as the expansion and contraction of its metal caused by changes in temperature.

  5. Contribution of V(H) replacement products in mouse antibody repertoire.

    PubMed

    Huang, Lin; Lange, Miles D; Yu, Yangsheng; Li, Song; Su, Kaihong; Zhang, Zhixin

    2013-01-01

    VH replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS) near the 3' end of a rearranged VH gene and the 23-bp RSS from an upstream unrearranged VH gene. Due to the location of the cRSS, VH replacement leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction, which can be used as a marker to identify VH replacement products. To determine the contribution of VH replacement products to mouse antibody repertoire, we developed a Java-based VH Replacement Footprint Analyzer (VHRFA) program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify VH replacement products. The overall frequency of VH replacement products in these IgH genes is 5.29% based on the identification of pentameric VH replacement footprints at their V-D junctions. The identified VH replacement products are distributed similarly in IgH genes using most families of VH genes, although different families of VH genes are used differentially. The frequencies of VH replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified VH replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.

  6. VH replacement in primary immunoglobulin repertoire diversification.

    PubMed

    Sun, Amy; Novobrantseva, Tatiana I; Coffre, Maryaline; Hewitt, Susannah L; Jensen, Kari; Skok, Jane A; Rajewsky, Klaus; Koralov, Sergei B

    2015-02-03

    The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. VH replacement represents a RAG-mediated secondary rearrangement in which an upstream VH element recombines with a rearranged VHDHJH joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original VH element and the conventional RSS of the invading VH gene, leaving behind a footprint of up to five base pairs (bps) of the original VH gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that VH replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive VHDHJH allele. Using this mouse model, we characterized the role of VH replacement in the diversification of the primary Ig repertoire through the modification of productive VHDHJH rearrangements. Our results indicate that VH replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies.

  7. Single implant tooth replacement.

    PubMed

    Briley, T F

    1998-01-01

    It has been shown that direct bone anchorage of dental implants will provide long-term predictability for single tooth implants and multi-unit implants. The function of implant-supported restoration is now routinely achieved. The real challenge facing the restorative dentist and laboratory technician is to achieve optimal aesthetics. The learning objective of this article is to review the prosthodontic procedures essential to maximizing natural aesthetics in implant supported restorations. It will provide a review of master impression techniques, prepable titanium abutments and designing the cement on restoration. Particular emphasis is directed to the soft tissue model from which a series of sequenced techniques can be followed to achieve optimal aesthetics. Analysis of the implant alignment with regard to the neighboring teeth will result in having to make a choice of which prepable abutment will maximize the aesthetic result. The following case outlines how to replace a single missing tooth using an externally hexed implant system and a prefabricated titanium abutment on a 26-year-old male patient.

  8. Reuse, replace, recycle

    PubMed Central

    McMurray, Michael A.; Thorner, Jeremy W.

    2015-01-01

    Septins are guanine nucleotide-binding proteins that form hetero-oligomeric complexes, which assemble into filaments and higher-order structures at sites of cell division and morphogenesis in eukaryotes. Dynamic changes in the organization of septin-containing structures occur concomitantly with progression through the mitotic cell cycle and during cell differentiation. Septins also undergo stage-specific post-translational modifications, which have been implicated in regulating their dynamics, in some cases via purported effects on septin turnover. In our recent study, the fate of two of the five septins expressed in mitotic cells of budding yeast (Saccharomyces cerevisiae) was tracked using two complementary fluorescence-based methods for pulse-chase analysis. During mitotic growth, previously-made molecules of both septins (Cdc10 and Cdc12) persisted through multiple successive divisions and were incorporated equivalently with newly synthesized molecules into hetero-oligomers and higher-order structures. Similarly, in cells undergoing meiosis and the developmental program of sporulation, pre-existing copies of Cdc10 were incorporated into new structures. In marked contrast, Cdc12 was irreversibly excluded from septin complexes and replaced by another septin, Spr3. Here, we discuss the broader implications of these results and related findings with regard to how septin dynamics is coordinated with the mitotic cell cycle and in the yeast life cycle, and how these observations may relate to control of the dynamics of other complex multi-subunit assemblies. PMID:19164941

  9. Hormone replacement therapy.

    PubMed

    Scharbo-Dehaan, M

    1996-12-01

    More than 40 million women in the United States are now going through or are past menopause. Another 3.5 million or more will reach midlife in the next decade. As their life expectancy increases (mean life expectancy of women is now approximately 84 years), so does the need for therapeutic regimens related to reproductive function and aging in woman. Few medical treatments available to menopausal and postmenopausal women have as much potential benefit as well as possible health risks as hormone replacement therapy (HRT). Despite the increasing amount of scientific data available regarding the benefits of HRT, a degree of uncertainty still remains, both in the minds of some women, and with some health professionals, regarding the risks associated with long-term therapy. Even though the literature is voluminous, contradictory, and unclear, health providers must be able to keep abreast of current knowledge about the benefits, risks, and unknowns of these drugs. The purpose of this article is to provide a review and an update on the types of hormones available for HRT, their pharmacology and pharmacokinetics, and their risks, benefits, and contraindications. Newer products, specially compounded formulas, new regimens, and new modes of delivery that offer women alternatives and allow care to be individualized are described. In addition, some of the ongoing management dilemmas that practitioners face with the woman who chooses HRT are presented with practical solutions and suggestions.

  10. Antenna Controller Replacement Software

    NASA Technical Reports Server (NTRS)

    Chao, Roger Y.; Morgan, Scott C.; Strain, Martha M.; Rockwell, Stephen T.; Shimizu, Kenneth J.; Tehrani, Barzia J.; Kwok, Jaclyn H.; Tuazon-Wong, Michelle; Valtier, Henry; Nalbandi, Reza; Wert, Michael; Leung, Patrick

    2010-01-01

    The Antenna Controller Replacement (ACR) software accurately points and monitors the Deep Space Network (DSN) 70-m and 34-m high-efficiency (HEF) ground-based antennas that are used to track primarily spacecraft and, periodically, celestial targets. To track a spacecraft, or other targets, the antenna must be accurately pointed at the spacecraft, which can be very far away with very weak signals. ACR s conical scanning capability collects the signal in a circular pattern around the target, calculates the location of the strongest signal, and adjusts the antenna pointing to point directly at the spacecraft. A real-time, closed-loop servo control algorithm performed every 0.02 second allows accurate positioning of the antenna in order to track these distant spacecraft. Additionally, this advanced servo control algorithm provides better antenna pointing performance in windy conditions. The ACR software provides high-level commands that provide a very easy user interface for the DSN operator. The operator only needs to enter two commands to start the antenna and subreflector, and Master Equatorial tracking. The most accurate antenna pointing is accomplished by aligning the antenna to the Master Equatorial, which because of its small size and sheltered location, has the most stable pointing. The antenna has hundreds of digital and analog monitor points. The ACR software provides compact displays to summarize the status of the antenna, subreflector, and the Master Equatorial. The ACR software has two major functions. First, it performs all of the steps required to accurately point the antenna (and subreflector and Master Equatorial) at the spacecraft (or celestial target). This involves controlling the antenna/ subreflector/Master-Equatorial hardware, initiating and monitoring the correct sequence of operations, calculating the position of the spacecraft relative to the antenna, executing the real-time servo control algorithm to maintain the correct position, and

  11. Gene Therapy

    PubMed Central

    Scheller, E.L.; Krebsbach, P.H.

    2009-01-01

    Gene therapy is defined as the treatment of disease by transfer of genetic material into cells. This review will explore methods available for gene transfer as well as current and potential applications for craniofacial regeneration, with emphasis on future development and design. Though non-viral gene delivery methods are limited by low gene transfer efficiency, they benefit from relative safety, low immunogenicity, ease of manufacture, and lack of DNA insert size limitation. In contrast, viral vectors are nature’s gene delivery machines that can be optimized to allow for tissue-specific targeting, site-specific chromosomal integration, and efficient long-term infection of dividing and non-dividing cells. In contrast to traditional replacement gene therapy, craniofacial regeneration seeks to use genetic vectors as supplemental building blocks for tissue growth and repair. Synergistic combination of viral gene therapy with craniofacial tissue engineering will significantly enhance our ability to repair and replace tissues in vivo. PMID:19641145

  12. Using your shoulder after replacement surgery

    MedlinePlus

    Joint replacement surgery - using your shoulder; Shoulder replacement surgery - after ... You have had shoulder replacement surgery to replace the bones of your shoulder joint with artificial parts. The parts include a stem made of metal and a ...

  13. Educating My Replacement

    NASA Astrophysics Data System (ADS)

    Tarter, Jill

    , in partnership with the dedicated teachers out there, I think I can help promote the critical thinking skills and scientific literacy of the next generation of voters. Hopefully, I can also help train my replacement to be a better scientist, capable of seizing all the opportunities generated by advances in technology and our improved understanding of the universe to craft search strategies with greater probability of success than those I have initiated.

  14. Genes

    MedlinePlus

    ... Search Search MedlinePlus GO GO About MedlinePlus Site Map FAQs Customer Support Health Topics Drugs & Supplements Videos & Tools Español You Are Here: Home → Medical Encyclopedia → Genes URL of this page: //medlineplus.gov/ency/article/ ...

  15. [Ascending aorta replacement late after aortic valve replacement].

    PubMed

    Hayashi, Yasunari; Ito, Toshiaki; Maekawa, Atsuo; Sawaki, Sadanari; Fujii, Genyo; Hoshino, Satoshi; Tokoro, Masayoshi; Yanagisawa, Junji

    2013-07-01

    Replacement of the asceding aorta is indicated in patients undergoing aortic valve replacement( AVR), if the diameter of the ascending aorta is greater than 5.0 cm. If the diameter of the asceding aorta is from 4.0 to 5.0 cm, it was arguable whether replacement of the ascending aorta should be performed. Nine patients who underwent reoperative ascending aorta replacement after AVR were reviewed retrospectively. Reoperation on the asending aorta replacement was performed 11.8±7.2 years (range 1y5m~23y3m) after AVR. Mean patient age was 69.9±6.3 (range 60~81). In 2 cases, reoperations were performed early year after AVR. Although ascending aorta was dilated at the 1st operation, replacement wasn't performed for the age and minimally invasive cardiac surgery (MICS). In 3 cases, reoperations were performed more than 10 years later. On these cases, ascending aorta aneurysm and dissection occurred with no pain and were pointed out by computed tomography(CT) or ultrasonic cardiogram(UCG). We think that patients with dilatation of the ascending aorta should undergo AVR and aorta replacement at the 1st operation regardness of age. It is important that patients who underwent AVR should undergo a regular checkup on the ascending aorta.

  16. Genetic method to analyze essential genes of Escherichia coli.

    PubMed

    Hupert-Kocurek, Katarzyna; Sage, Jay M; Makowska-Grzyska, Magdalena; Kaguni, Jon M

    2007-11-01

    The genetic analysis of essential genes has been generally restricted to the use of conditional mutations, or inactivating chromosomal mutations, which require a complementing plasmid that must either be counterselected or lost to measure a phenotype. These approaches are limited because they do not permit the analysis of mutations suspected to affect a specific function of a protein, nor do they take advantage of the increasing abundance of structural and bioinformatics data for proteins. Using the dnaC gene as an example, we developed a genetic method that should permit the mutational analysis of other essential genes of Escherichia coli and related enterobacteria. The method consists of using a strain carrying a large deletion of the dnaC gene, which is complemented by a wild-type copy expressed from a plasmid that requires isopropyl-beta-d-thiogalactopyranoside for maintenance. Under conditions in which this resident plasmid is lost, the method measures the function of a dnaC mutation encoded by a second plasmid. This methodology should be widely applicable to the genetic analysis of other essential genes.

  17. The role of the dental lamina in mammalian tooth replacement.

    PubMed

    Järvinen, Elina; Tummers, Mark; Thesleff, Irma

    2009-06-15

    We have applied the ferret, Mustela putorius furo, as a model for tooth replacement. Ferret has a heterodont dentition, which includes all tooth families, and all antemolar teeth are replaced. Compared with mouse, the ferret therefore has a less derived mammalian dentition resembling that of humans. We have studied tooth replacement in serial histological sections in embryonic and young postnatal ferrets. Our observations indicate that the replacement teeth form from the dental lamina that is intimately connected to the lingual aspect of the deciduous tooth enamel organ. It grows as an offshoot from the enamel organ, elongates in cervical direction and later buds to give rise to the replacement tooth. The extent of the dental lamina growth, preceding replacement tooth budding, varied between different teeth. The dynamic gene expression patterns of Sostdc1, Shh and Axin2 brought new insight into the signal networks regulating the tooth replacement process. The distinct expression of Sostdc1 at the interface between the dental lamina and the deciduous tooth is the first indication of a specific tissue identity of the dental lamina. We suggest that the reactivation of a competent dental lamina is pivotal for the replacement tooth formation. (c) 2009 Wiley-Liss, Inc.

  18. Fully functional bioengineered tooth replacement as an organ replacement therapy

    PubMed Central

    Ikeda, Etsuko; Morita, Ritsuko; Nakao, Kazuhisa; Ishida, Kentaro; Nakamura, Takashi; Takano-Yamamoto, Teruko; Ogawa, Miho; Mizuno, Mitsumasa; Kasugai, Shohei; Tsuji, Takashi

    2009-01-01

    Current approaches to the development of regenerative therapies have been influenced by our understanding of embryonic development, stem cell biology, and tissue engineering technology. The ultimate goal of regenerative therapy is to develop fully functioning bioengineered organs which work in cooperation with surrounding tissues to replace organs that were lost or damaged as a result of disease, injury, or aging. Here, we report a successful fully functioning tooth replacement in an adult mouse achieved through the transplantation of bioengineered tooth germ into the alveolar bone in the lost tooth region. We propose this technology as a model for future organ replacement therapies. The bioengineered tooth, which was erupted and occluded, had the correct tooth structure, hardness of mineralized tissues for mastication, and response to noxious stimulations such as mechanical stress and pain in cooperation with other oral and maxillofacial tissues. This study represents a substantial advance and emphasizes the potential for bioengineered organ replacement in future regenerative therapies. PMID:19666587

  19. Fully functional bioengineered tooth replacement as an organ replacement therapy.

    PubMed

    Ikeda, Etsuko; Morita, Ritsuko; Nakao, Kazuhisa; Ishida, Kentaro; Nakamura, Takashi; Takano-Yamamoto, Teruko; Ogawa, Miho; Mizuno, Mitsumasa; Kasugai, Shohei; Tsuji, Takashi

    2009-08-11

    Current approaches to the development of regenerative therapies have been influenced by our understanding of embryonic development, stem cell biology, and tissue engineering technology. The ultimate goal of regenerative therapy is to develop fully functioning bioengineered organs which work in cooperation with surrounding tissues to replace organs that were lost or damaged as a result of disease, injury, or aging. Here, we report a successful fully functioning tooth replacement in an adult mouse achieved through the transplantation of bioengineered tooth germ into the alveolar bone in the lost tooth region. We propose this technology as a model for future organ replacement therapies. The bioengineered tooth, which was erupted and occluded, had the correct tooth structure, hardness of mineralized tissues for mastication, and response to noxious stimulations such as mechanical stress and pain in cooperation with other oral and maxillofacial tissues. This study represents a substantial advance and emphasizes the potential for bioengineered organ replacement in future regenerative therapies.

  20. INNOPLANT Total Hip Replacement System.

    PubMed

    Harper, Tisha A M

    2017-07-01

    Total hip replacement is a salvage procedure that is done to alleviate discomfort secondary to osteoarthritis in the hip, which is most often a result of hip dysplasia. Commercially available total hip replacement implants for small animal patients are classified as cemented or cementless. The INNOPLANT Total Hip Replacement system includes modular, screw-in cementless components that were developed to improve implant stability by maintaining as much normal anatomic structure, and by extension biomechanics of the coxofemoral joint, as possible. As a newer system, there are few data and no long-term studies available in the veterinary literature. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Genome engineering and gene expression control for bacterial strain development.

    PubMed

    Song, Chan Woo; Lee, Joungmin; Lee, Sang Yup

    2015-01-01

    In recent years, a number of techniques and tools have been developed for genome engineering and gene expression control to achieve desired phenotypes of various bacteria. Here we review and discuss the recent advances in bacterial genome manipulation and gene expression control techniques, and their actual uses with accompanying examples. Genome engineering has been commonly performed based on homologous recombination. During such genome manipulation, the counterselection systems employing SacB or nucleases have mainly been used for the efficient selection of desired engineered strains. The recombineering technology enables simple and more rapid manipulation of the bacterial genome. The group II intron-mediated genome engineering technology is another option for some bacteria that are difficult to be engineered by homologous recombination. Due to the increasing demands on high-throughput screening of bacterial strains having the desired phenotypes, several multiplex genome engineering techniques have recently been developed and validated in some bacteria. Another approach to achieve desired bacterial phenotypes is the repression of target gene expression without the modification of genome sequences. This can be performed by expressing antisense RNA, small regulatory RNA, or CRISPR RNA to repress target gene expression at the transcriptional or translational level. All of these techniques allow efficient and rapid development and screening of bacterial strains having desired phenotypes, and more advanced techniques are expected to be seen. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Fracture After Total Hip Replacement

    MedlinePlus

    ... er Total Hip Replacement cont. • Dislocation • Limb length inequality • Poor fracture healing • Repeat fracture • Lack of in- ... Surgeons (AAOS). To learn more about your orthopaedic health, please visit orthoinfo.org. Page ( 5 ) AAOS does ...

  3. An encephalocele replacing nose completely.

    PubMed

    Dasgupta, G

    1999-01-01

    Congenital encephalocele presenting in the nasal region is extremely rare. We present here a case ot tncephaloce lecompletely replacing the nose of a five year old boy and duscuss about similar swellings in this region.

  4. What Is a Hip Replacement?

    MedlinePlus

    ... reduce joint pain and stiffness. It can increase muscle strength and joint range of motion (how much you ... goals after hip replacement surgery are to: Increase muscle strength Increase your blood circulation and overall fitness Avoid ...

  5. Single Project Replaces District's Windows.

    ERIC Educational Resources Information Center

    American School and University, 1981

    1981-01-01

    The towns of Maplewood and South Orange (New Jersey) earmarked over $1 million, raised from a bond issue, to replace with thermal aluminum-framed units the 2400 worn out windows in eight 50-year-old schools. (Author)

  6. The development and testing of new nicotine replacement treatments: from 'nicotine replacement' to 'smoking replacement'.

    PubMed

    Hajek, Peter

    2015-07-01

    Griffith Edwards, unusually in the 1970s, saw tobacco use as falling within the remit of addiction research, and brought Michael Russell to the Addiction Research Unit [ARU] to initiate research into smoking. The work of the tobacco section of ARU paved the way to a better understanding of tobacco dependence and to developing nicotine replacement treatments. Michael Russell pioneered the idea of attractive nicotine replacement products with an acceptable safety profile replacing cigarettes on the open market and ending the tobacco epidemic, envisaging a transition from medicinal and temporary 'nicotine replacement' to recreational and potentially permanent 'smoking replacement'. Mike's prediction that the pharmaceutical industry would develop such devices did not materialize. Instead, two such products were generated by the tobacco industry (snus) and independent developers (electronic cigarettes). Another of Mike's hopes was that regulators would adopt rational policies, and that tobacco control activists would become supportive of smoking replacement once they thought through the implications. Until now, the 'smoking replacement' idea has been met with vigorous opposition from some tobacco control activists. The voices of researchers with historical links to ARU are prominent in arguing in favour of harm reduction and e-cigarettes. The most important debate ever to occur in tobacco control is under way and it carries the signature of Griffith Edwards' ARU. © 2015 Society for the Study of Addiction.

  7. Prioritization Methodology for Chemical Replacement

    NASA Technical Reports Server (NTRS)

    Cruit, W.; Schutzenhofer, S.; Goldberg, B.; Everhart, K.

    1993-01-01

    This project serves to define an appropriate methodology for effective prioritization of efforts required to develop replacement technologies mandated by imposed and forecast legislation. The methodology used is a semiquantitative approach derived from quality function deployment techniques (QFD Matrix). This methodology aims to weigh the full environmental, cost, safety, reliability, and programmatic implications of replacement technology development to allow appropriate identification of viable candidates and programmatic alternatives. The results are being implemented as a guideline for consideration for current NASA propulsion systems.

  8. Potential Functional Replacement of the Plastidic Acetyl-CoA Carboxylase Subunit (accD) Gene by Recent Transfers to the Nucleus in Some Angiosperm Lineages1[W][OA

    PubMed Central

    Rousseau-Gueutin, Mathieu; Huang, Xun; Higginson, Emily; Ayliffe, Michael; Day, Anil; Timmis, Jeremy N.

    2013-01-01

    Eukaryotic cells originated when an ancestor of the nucleated cell engulfed bacterial endosymbionts that gradually evolved into the mitochondrion and the chloroplast. Soon after these endosymbiotic events, thousands of ancestral prokaryotic genes were functionally transferred from the endosymbionts to the nucleus. This process of functional gene relocation, now rare in eukaryotes, continues in angiosperms. In this article, we show that the chloroplastic acetyl-CoA carboxylase subunit (accD) gene that is present in the plastome of most angiosperms has been functionally relocated to the nucleus in the Campanulaceae. Surprisingly, the nucleus-encoded accD transcript is considerably smaller than the plastidic version, consisting of little more than the carboxylase domain of the plastidic accD gene fused to a coding region encoding a plastid targeting peptide. We verified experimentally the presence of a chloroplastic transit peptide by showing that the product of the nuclear accD fused to green fluorescent protein was imported in the chloroplasts. The nuclear gene regulatory elements that enabled the erstwhile plastidic gene to become functional in the nuclear genome were identified, and the evolution of the intronic and exonic sequences in the nucleus is described. Relocation and truncation of the accD gene is a remarkable example of the processes underpinning endosymbiotic evolution. PMID:23435694

  9. Prioritization methodology for chemical replacement

    NASA Technical Reports Server (NTRS)

    Cruit, Wendy; Goldberg, Ben; Schutzenhofer, Scott

    1995-01-01

    Since United States of America federal legislation has required ozone depleting chemicals (class 1 & 2) to be banned from production, The National Aeronautics and Space Administration (NASA) and industry have been required to find other chemicals and methods to replace these target chemicals. This project was initiated as a development of a prioritization methodology suitable for assessing and ranking existing processes for replacement 'urgency.' The methodology was produced in the form of a workbook (NASA Technical Paper 3421). The final workbook contains two tools, one for evaluation and one for prioritization. The two tools are interconnected in that they were developed from one central theme - chemical replacement due to imposed laws and regulations. This workbook provides matrices, detailed explanations of how to use them, and a detailed methodology for prioritization of replacement technology. The main objective is to provide a GUIDELINE to help direct the research for replacement technology. The approach for prioritization called for a system which would result in a numerical rating for the chemicals and processes being assessed. A Quality Function Deployment (QFD) technique was used in order to determine numerical values which would correspond to the concerns raised and their respective importance to the process. This workbook defines the approach and the application of the QFD matrix. This technique: (1) provides a standard database for technology that can be easily reviewed, and (2) provides a standard format for information when requesting resources for further research for chemical replacement technology. Originally, this workbook was to be used for Class 1 and Class 2 chemicals, but it was specifically designed to be flexible enough to be used for any chemical used in a process (if the chemical and/or process needs to be replaced). The methodology consists of comparison matrices (and the smaller comparison components) which allow replacement technology

  10. Optimization of station battery replacement

    NASA Astrophysics Data System (ADS)

    Jancauskas, J. R.; Shook, D. A.

    1994-08-01

    During a loss of ac power at a nuclear generating station (including diesel generators), batteries provide the source of power which is required to operate safety-related components. Because traditional lead-acid batteries have a qualified life of 20 years, the batteries must be replaced a minimum of once during a station's lifetime, twice if license extension is pursued, and more often depending on actual in-service dates and the results of surveillance tests. Replacement of batteries often occurs prior to 20 years as a result of systems changes caused by factors such as Station Blackout Regulations, control system upgrades, incremental load growth, and changes in the operating times of existing equipment. Many of these replacement decisions are based on the predictive capabilities of manual design basis calculations. The inherent conservatism of manual calculations may result in battery replacements occurring before actually required. Computerized analysis of batteries can aid in optimizing the timing of replacements as well as in interpreting service test data. Computerized analysis also provides large benefits in maintaining the as-configured load profile and corresponding design margins, while also providing the capability to quickly analyze proposed modifications and respond to internal and external audits.

  11. Nonclinical Development of BCG Replacement Vaccine Candidates.

    PubMed

    Velmurugan, Kamalakannan; Grode, Leander; Chang, Rosemary; Fitzpatrick, Megan; Laddy, Dominick; Hokey, David; Derrick, Steven; Morris, Sheldon; McCown, David; Kidd, Reginald; Gengenbacher, Martin; Eisele, Bernd; Kaufmann, Stefan H E; Fulkerson, John; Brennan, Michael J

    2013-04-16

    The failure of current Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccines, given to neonates to protect against adult tuberculosis and the risk of using these live vaccines in HIV-infected infants, has emphasized the need for generating new, more efficacious and safer replacement vaccines. With the availability of genetic techniques for constructing recombinant BCG (rBCG) strains containing well-defined gene deletions or insertions, new vaccine candidates are under evaluation at both the preclinical and clinical stages of development. Since most BCG vaccines in use today were evaluated in clinical trials decades ago and are produced by outdated processes, the development of new BCG vaccines offers a number of advantages that include a modern well-defined manufacturing process along with state-of-the-art evaluation of safety and efficacy in target populations. We provide a description of the preclinical development of two novel rBCGs, VPM1002 that was constructed by adding a modified hly gene coding for the protein listeriolysin O (LLO) from Listeria monocytogenes and AERAS-422, which carries a modified pfoA gene coding for the protein perfringolysin O (PFO) from Clostridium perfringens, and three genes from Mycobacterium tuberculosis. Novel approaches like these should be helpful in generating stable and effective rBCG vaccine candidates that can be better characterized than traditional BCG vaccines.

  12. [Hip replacement in military personnel].

    PubMed

    Maksimov, I B; Paniushin, K A; Brizhan', L K; Buriachenko, B P; Varfolomeev, D I; Mimanchev, O V

    2014-01-01

    Hip joint diseases and injuries are common for orthopedic pathology among military personnel. Hip replacement is one of the most frequent operations. Authors evaluated hip replacement in 136 servicemen treated at the center of traumatology and orthopedics of Burdenko General Military Clinical Hospital of the Ministry of Defense of Russia in 2010-2013. On the basis of the conducted analysis the main disease groups were revealed, peculiarities of pathology among this category of patients. Authors proposed surgical doctrine for the treatment of this contingent. Effective surgical treatment, in particular, hip replacement surgery, conducted with the use of the proposed principles, as a rule, fully functionally cured patients and contributes to return them in the system that contributes to the strengthening of defensibility of the country.

  13. Prioritization methodology for chemical replacement

    NASA Technical Reports Server (NTRS)

    Goldberg, Ben; Cruit, Wendy; Schutzenhofer, Scott

    1995-01-01

    This methodology serves to define a system for effective prioritization of efforts required to develop replacement technologies mandated by imposed and forecast legislation. The methodology used is a semi quantitative approach derived from quality function deployment techniques (QFD Matrix). QFD is a conceptual map that provides a method of transforming customer wants and needs into quantitative engineering terms. This methodology aims to weight the full environmental, cost, safety, reliability, and programmatic implications of replacement technology development to allow appropriate identification of viable candidates and programmatic alternatives.

  14. [Nicotine replacement therapy during pregnancy].

    PubMed

    Jiménez Ruiz, Carlos A

    2006-08-01

    In spite of the fact that smoking is the main cause of obstetric morbidity and mortality, 20% of North American women smoke during pregnancy. In Spain, this figure is as high as 30% to 35%. Treating smoking addiction in these patients should be the first and most important therapeutic measure undertaken by health professionals responsible for the care of pregnant women. All experts recommend the use of nicotine replacement therapy when more conservative treatments have failed. This article reviews the indications and contraindications for nicotine replacement therapy during pregnancy as well as other issues relating to the safety of this type of treatment.

  15. Prioritization methodology for chemical replacement

    NASA Technical Reports Server (NTRS)

    Goldberg, Ben; Cruit, Wendy; Schutzenhofer, Scott

    1995-01-01

    This methodology serves to define a system for effective prioritization of efforts required to develop replacement technologies mandated by imposed and forecast legislation. The methodology used is a semi quantitative approach derived from quality function deployment techniques (QFD Matrix). QFD is a conceptual map that provides a method of transforming customer wants and needs into quantitative engineering terms. This methodology aims to weight the full environmental, cost, safety, reliability, and programmatic implications of replacement technology development to allow appropriate identification of viable candidates and programmatic alternatives.

  16. HST Replacement Battery Initial Performance

    NASA Technical Reports Server (NTRS)

    Krol, Stan; Waldo, Greg; Hollandsworth, Roger

    2009-01-01

    The Hubble Space Telescope (HST) original Nickel-Hydrogen (NiH2) batteries were replaced during the Servicing Mission 4 (SM4) after 19 years and one month on orbit.The purpose of this presentation is to highlight the findings from the assessment of the initial sm4 replacement battery performance. The batteries are described, the 0 C capacity is reviewed, descriptions, charts and tables reviewing the State Of Charge (SOC) Performance, the Battery Voltage Performance, the battery impedance, the minimum voltage performance, the thermal performance, the battery current, and the battery system recharge ratio,

  17. Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

    PubMed Central

    2009-01-01

    Background Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. Results Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the λ-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6 × His, 3 × FLAG, 4 × ProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the λ-Red system, which can lead to unwanted secondary

  18. Cloning-independent markerless gene editing in Streptococcus sanguinis: novel insights in type IV pilus biology.

    PubMed

    Gurung, Ishwori; Berry, Jamie-Lee; Hall, Alexander M J; Pelicic, Vladimir

    2016-11-29

    Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)-exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags. We show that (i) the last six genes in the pil locus harbouring all the genes dedicated to Tfp biology play no role in piliation or Tfp-mediated motility, (ii) two highly conserved Asp residues are crucial for enzymatic activity of the prepilin peptidase PilD and (iii) that pilin subunits with a C-terminally appended hexa-histidine (6His) tag are still assembled into functional Tfp. The methodology for genetic manipulation we describe here should be broadly applicable.

  19. Replaceable Sleeve Protects Welder Coil

    NASA Technical Reports Server (NTRS)

    Baker, W. L.; Simpson, C., E.

    1983-01-01

    New replaceable carbon insert for deflection coil in electron-beam welder promises to decrease maintenance costs. Inserts made from materials other than carbon (not yet tried) are less expensive, thus reducing costs even further. With carbon insert, deflection coils last longer and are easier to maintain.

  20. Testing of Replacement Bag Material

    SciTech Connect

    Laurinat, J.E.

    1998-11-03

    Recently, the FB-Line bagout material was changed to simplify the processing of sand, slag, and crucible.The results of the strength tests and the outgassing measurements and calculations demonstrate that the proposed replacement nylon bag materials (HRMP and orange anti-static material) are acceptable substitutes for LDPE and the original nylon with respect to mechanical properties.

  1. Will peanut hulls replace oil

    SciTech Connect

    Not Available

    1980-12-01

    A low-cost, fast-curing wood adhesive has been recently developed by the University of Georgia, in which up to 80% of the petroleum ingredients can be replaced with a substance extracted from peanut hulls. An outline of the process is given.

  2. Replaceable Sleeve Protects Welder Coil

    NASA Technical Reports Server (NTRS)

    Baker, W. L.; Simpson, C., E.

    1983-01-01

    New replaceable carbon insert for deflection coil in electron-beam welder promises to decrease maintenance costs. Inserts made from materials other than carbon (not yet tried) are less expensive, thus reducing costs even further. With carbon insert, deflection coils last longer and are easier to maintain.

  3. MELFI-3 Electronic Unit Replacement

    NASA Image and Video Library

    2011-07-01

    ISS028-E-013797 (1 July 2011) --- NASA astronaut Mike Fossum, Expedition 28 flight engineer, replaces a failed electronic unit for the Minus Eighty Laboratory Freezer for ISS 3 (MELFI-3) with a spare unit in the Kibo laboratory of the International Space Station.

  4. MELFI-3 Electronic Unit Replacement

    NASA Image and Video Library

    2011-07-01

    ISS028-E-013799 (1 July 2011) --- NASA astronaut Mike Fossum, Expedition 28 flight engineer, replaces a failed electronic unit for the Minus Eighty Laboratory Freezer for ISS 3 (MELFI-3) with a spare unit in the Kibo laboratory of the International Space Station.

  5. Bonding over Dentin Replacement Materials.

    PubMed

    Meraji, Naghmeh; Camilleri, Josette

    2017-08-01

    Dentin replacement materials are necessary in large cavities to protect the pulp and reduce the bulk of filling material. These materials are layered with a composite resin restorative material. Microleakage caused by poor bonding of composite resin to underlying dentin replacement material will result in pulp damage. The aim of this study was to characterize the interface between dentin replacement materials and composite resin and to measure the shear bond strength after dynamic aging. Biodentine (Septodont, Saint Maur-des-Fosses, France), Theracal LC (Bisco, Schaumburg, IL), and Fuji IX (GC, Tokyo, Japan) were used as dentin replacement materials. They were then overlaid with a total-etch and bonding agent or a self-etch primer and composite resin or a glass ionomer cement. All combinations were thermocycled for 3000 cycles. The interface was characterized using scanning electron microscopy and elemental mapping. Furthermore, the shear bond strength was assessed. The Biodentine surface was modified by etching. The Theracal LC and Fuji IX microstructure was unchanged upon the application of acid etch. The Biodentine and glass ionomer interface showed an evident wide open space, and glass particles from the glass ionomer adhered to the Biodentine surface. Elemental migration was shown with aluminum, barium, fluorine, and ytterbium present in Biodentine from the overlying composite resin. Calcium was more stable. The bond strength between Theracal LC and composite using a total-etch technique followed by self-etch primer achieved the best bond strength values. Biodentine exhibited the weakest bond with complete failure of bonding shown after demolding and thermocycling. Dynamic aging is necessary to have clinically valid data. Bonding composite resin to water-based dentin replacement materials is still challenging, and further alternatives for restoration of teeth using such materials need to be developed. Copyright © 2017 American Association of Endodontists

  6. Pseudoaneurysm after ICD battery replacement.

    PubMed

    Schulz, S L; Lehmkuhl, H; Zipfel, K B; Hetzer, R

    2007-02-01

    False aneurysms of the extracranial carotid arteries are rare and mainly of traumatic origin. We report on a patient who presented with a sudden onset mass in his right neck 2 weeks after routine replacement of his ICD battery. He had received systemic anticoagulation since an aortic valve replacement 5 years before. By color Doppler sonography the mass was identified as a partly thrombosed false aneurysm originating from the common carotid artery. As no spontaneous resolution occurred during the following days the aneurysm was removed surgically 5 days later without further complications. However no connection to the common carotid artery was found at surgery. Yet in the histopathological examination the specimen showed the morphological characteristics of a pseudoaneurysm. There was no history of neck-trauma and no attempted vascular access during the recent operation.

  7. [MINIMALLY INVASIVE AORTIC VALVE REPLACEMENT].

    PubMed

    Tabata, Minoru

    2016-03-01

    Minimally invasive aortic valve replacement (MIAVR) is defined as aortic valve replacement avoiding full sternotomy. Common approaches include a partial sternotomy right thoracotomy, and a parasternal approach. MIAVR has been shown to have advantages over conventional AVR such as shorter length of stay and smaller amount of blood transfusion and better cosmesis. However, it is also known to have disadvantages such as longer cardiopulmonary bypass and aortic cross-clamp times and potential complications related to peripheral cannulation. Appropriate patient selection is very important. Since the procedure is more complex than conventional AVR, more intensive teamwork in the operating room is essential. Additionally, a team approach during postoperative management is critical to maximize the benefits of MIAVR.

  8. Hurricane intensity and eyewall replacement.

    PubMed

    Houze, Robert A; Chen, Shuyi S; Smull, Bradley F; Lee, Wen-Chau; Bell, Michael M

    2007-03-02

    Observations made during the historic 2005 hurricane season document a case of "eyewall replacement." Clouds outside the hurricane eyewall coalesce to form a new eyewall at a greater radius from the storm center, and the old eyewall dies. The winds in the new eyewall are initially weaker than those in the original eyewall, but as the new eyewall contracts, the storm reintensifies. Understanding this replacement mechanism is vital to forecasting variations in hurricane intensity. Processes in the "moat" region between the new and old eyewall have been particularly unclear. Aircraft data now show that the moat becomes dynamically similar to the eye and thus is converted into a region inimical to survival of the inner eyewall. We suggest that targeting aircraft to key parts of the storm to gain crucial input to high-resolution numerical models can lead to improvements in forecasting hurricane intensity.

  9. Hinge total knee replacement revisited

    PubMed Central

    Cameron, Hugh U.; Hu, Cungen; Vyamont, Didier

    1997-01-01

    Objective To determine if aseptic loosening is a major problem in hinge total knee replacement. Design A cohort study. Setting A university-affiliated institute, specializing in elective orthopedic surgery. Patients Fifty-eight patients, mainly those requiring revision, in whom the conditions were such that it was felt only a totally constrained implant was appropriate. In 7 patients the implant was press-fitted; in the remainder it was cemented. Five patients required fusion or revision, and 8 died less than 2 years after implantation, leaving 45 for review. Follow-up was 2 to 13 years. Intervention Total knee replacement with a Guepar II prosthesis. Main outcome measures Radiolucency determined by the Cameron system and clinical scoring using the Hospital for Special Surgery system. Results Of the cemented components, 91% of femoral stems were type IA (no lucency), 9% were type IB (partial lucency), with no type II or III lucency. Tibial lucency was 87% type IA and 13% type IB, with no type II or III lucency. Of the noncemented components, 58% of femoral components were type IA and 42% type IB. Tibial lucency was 71% type IA and 29% type IB. Lucency was mainly present in zones 1 and 2 adjacent to the knee. Clinical rating was 18% excellent, 20% good, 20% fair and 42% poor. Postoperative complications included infection (13%), aseptic loosening (7%), quadriceps lag (16%) and extensor mechanism problems (16%). Conclusions Aseptic loosening is an uncommon problem in hinge total knee replacement. The complication rate in cases of sufficient severity as to require a hinge replacement remains high. Current indications for a hinge prosthesis are anteroposterior instability with a very large flexion gap, complete absence of the collateral ligaments and complete absence of a functioning extensor mechanism. PMID:9267296

  10. Nicotine Replacement Therapy: An Overview

    PubMed Central

    Wadgave, Umesh; Nagesh, L

    2016-01-01

    Today tobacco use is the single greatest preventable cause of death in the world. Tobacco use is often incorrectly perceived to be solely a personal choice. This is contradicted by the fact that when fully aware of the health impact, most tobacco users want to quit but find it difficult to stop due to the addictiveness of nicotine. Henceforth, Nicotine replacement therapy (NRT) came into existence which temporarily replaces much of the nicotine from tobacco to reduce motivation to consume tobacco and nicotine withdrawal symptoms, thus easing the transition from cigarette smoking to complete abstinence. Various alternative nicotine sources (gum, transdermal patch, nasal spray, inhaler and sublingual tablets/lozenges) have been incorporated into tobacco cessation programs. Recent research is more focusing on rapid delivery of nicotine (Nicotine preloading, true pulmonary inhaler) and immunological approaches (nicotine vaccine) to tackle nicotine dependence. These NRTs are in general well tolerated and have minimal adverse effects. The review aims to summarize literature on various modes of nicotine replacement therapy methods currently used to treat nicotine dependence, and to give an overview about future possible approaches to treat tobacco use disorder. PMID:27610066

  11. The caudal septum replacement graft.

    PubMed

    Foda, Hossam M T

    2008-01-01

    To describe a technique for reconstructing the lost tip support in cases involving caudal septal and premaxillary deficiencies. The study included 120 patients with aesthetic and functional nasal problems resulting from the loss of caudal septal and premaxillary support. An external rhinoplasty approach was performed to reconstruct the lost support using a cartilaginous caudal septum replacement graft and premaxillary augmentation with Mersilene mesh. The majority of cases (75%) involved revisions in patients who had previously undergone 1 or more nasal surgical procedures. A caudal septum replacement graft was combined with premaxillary augmentation in 93 patients (77.5%). The mean follow-up period was 3 years (range, 1-12 years). The technique succeeded in correcting the external nasal deformities in all patients and resulted in a significant improvement in breathing in 74 patients (86%) with preoperative nasal obstruction. There were no cases of infection, displacement, or extrusion. The caudal septum replacement graft proved to be very effective in restoring the lost tip support in patients with caudal septal deficiency. Combining the graft with premaxillary augmentation using Mersilene mesh helped increase support and stability over long-term follow-up.

  12. Fusion following failed total ankle replacement.

    PubMed

    Wünschel, Markus; Leichtle, Ulf G; Leichtle, Carmen I; Walter, Christian; Mittag, Falk; Arlt, Eva; Suckel, Andreas

    2013-04-01

    Although mid- to long-term results after total ankle replacement have improved because of available second- and third-generation devices, failure of total ankle replacement is still more common compared with total hip replacement and total knee replacement. The portfolio of available total ankle replacement revision component options is small. Furthermore, the bone stock of the tibiotalar region is scarce making it difficult and in some situations impossible to perform revision total ankle replacement. In these cases tibiotalar and tibiotalocalcaneal fusions are valuable options. This article describes which surgical procedures should be performed depending on the initial situation and gives detailed advice on surgical technique, postoperative care, and clinical results.

  13. Gene doping.

    PubMed

    Azzazy, Hassan M E

    2010-01-01

    Gene doping abuses the legitimate approach of gene therapy. While gene therapy aims to correct genetic disorders by introducing a foreign gene to replace an existing faulty one or by manipulating existing gene(s) to achieve a therapeutic benefit, gene doping employs the same concepts to bestow performance advantages on athletes over their competitors. Recent developments in genetic engineering have contributed significantly to the progress of gene therapy research and currently numerous clinical trials are underway. Some athletes and their staff are probably watching this progress closely. Any gene that plays a role in muscle development, oxygen delivery to tissues, neuromuscular coordination, or even pain control is considered a candidate for gene dopers. Unfortunately, detecting gene doping is technically very difficult because the transgenic proteins expressed by the introduced genes are similar to their endogenous counterparts. Researchers today are racing the clock because assuring the continued integrity of sports competition depends on their ability to develop effective detection strategies in preparation for the 2012 Olympics, which may mark the appearance of genetically modified athletes.

  14. Dithizone staining of intracellular zinc: an unexpected and versatile counterscreen for auxotrophic marker genes in Saccharomyces cerevisiae.

    PubMed

    Yuan, Daniel S

    2011-01-01

    Auxotrophic marker genes such as URA3, LEU2, and HIS3 in Saccharomyces cerevisiae have long been used to select cells that have been successfully transformed with recombinant DNA. A longstanding challenge in working with these genes is that counterselection procedures are often lacking. This paper describes the unexpected discovery of a simple plate assay that imparts a bright red stain to cells experiencing nutritional stress from the lack of a marker gene. The procedure specifically stains a zinc-rich vesicular compartment analogous to the zinc-rich secretory vesicles found in insulin-secreting pancreatic islet cells and glutamate-secreting neurons. Staining was greatly diminished in zap1 mutants, which lack a homeostatic activator of zinc uptake, and in cot1 zrc1 double mutants, which lack the two yeast homologs of mammalian vesicle-specific zinc export proteins. Only one of 93 strains with temperature-sensitive alleles of essential genes exhibited an increase in dithizone staining at its non-permissive temperature, indicating that staining is not simply a sign of growth-arrested or dying cells. Remarkably, the procedure works with most commonly used marker genes, highlights subtle defects, uses no reporter constructs or expensive reagents, requires only a few hours of incubation, yields visually striking results without any instrumentation, and is not toxic to the cells. Many potential applications exist for dithizone staining, both as a versatile counterscreen for auxotrophic marker genes and as a powerful new tool for the genetic analysis of a biomedically important vesicular organelle.

  15. Robotically assisted mitral valve replacement.

    PubMed

    Gao, Changqing; Yang, Ming; Xiao, Cangsong; Wang, Gang; Wu, Yang; Wang, Jiali; Li, Jiachun

    2012-04-01

    In the present study, we determined the safety and efficacy of robotic mitral valve replacement using robotic technology. From January 2007 through March 2011, more than 400 patients underwent various types of robotic cardiac surgery in our department. Of these, 22 consecutive patients underwent robotically assisted mitral valve replacement. Of the 22 patients with isolated rheumatic mitral valve stenosis (9 men and 13 women), the mean age was 44.7 ± 19.8 years (range, 32-65). Preoperatively, all patients underwent a complete workup, including coronary angiography and transthoracic echocardiography. Of the 22 patients, 15 had concomitant atrial fibrillation. The surgical approach was through 4 right-side chest ports with femoral perfusion. Aortic occlusion was performed with a Chitwood crossclamp, and antegrade cardioplegia was administered directly by way of the anterior chest. Using 3 port incisions in the right side of the chest and a 2.5- to 3.0-cm working port, all the procedures were completed with the da Vinci S robot. All patients underwent successful robotic surgery. Of the 22 patients, 16 received a mechanical valve and 6 a tissue valve. The mean cardiopulmonary bypass time and aortic crossclamp time was 137.1 ± 21.9 minutes (range, 105-168) and 99.3 ± 17.9 minutes (range, 80-133), respectively. No operative deaths, stroke, or other complications occurred, and no incisional conversions were required. After surgery, all the patients were followed up echocardiographically. Robotically assisted mitral valve replacement can be performed safely in patients with isolated mitral valve stenosis, and surgical results are excellent. Copyright © 2012 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.

  16. Solvent replacement for green processing.

    PubMed Central

    Sherman, J; Chin, B; Huibers, P D; Garcia-Valls, R; Hatton, T A

    1998-01-01

    The implementation of the Montreal Protocol, the Clean Air Act, and the Pollution Prevention Act of 1990 has resulted in increased awareness of organic solvent use in chemical processing. The advances made in the search to find "green" replacements for traditional solvents are reviewed, with reference to solvent alternatives for cleaning, coatings, and chemical reaction and separation processes. The development of solvent databases and computational methods that aid in the selection and/or design of feasible or optimal environmentally benign solvent alternatives for specific applications is also discussed. Images Figure 2 Figure 3 PMID:9539018

  17. Senate committee recommends replacing FEMA

    NASA Astrophysics Data System (ADS)

    Zielinski, Sarah

    2006-05-01

    Flaws in the U.S. Federal Emergency Management Agency (FEMA) that were exposed after Hurricane Katrina struck the U.S. Gulf Coast in August 2005 are too substantial to be fixed, and the agency should be replaced with a more capable structure, recommends a 27 April report released by the Senate Committee on Homeland Security and Government Affairs. ``FEMA is discredited, demoralized, and dysfunctional. It is beyond repair. Just tweaking the organizational chart will not solve the problem,'' said Committee Chair Sen. Susan Collins (R-Maine).

  18. Liver Replacement for Pediatric Patients

    PubMed Central

    Starzl, Thomas E.; Koep, Lawrence J.; Schröter, Gerhard P. J.; Halgrimson, Charles G.; Porter, Kendrick A.; Weil, Richard

    2010-01-01

    Between March 1963 and January 1978, 74 patients 18 years of age or younger have had liver replacements at the University of Colorado Medical Center, Denver. The most common cause of native liver failure was biliary atresia (48/74, 65%); the second most common cause was chronic aggressive hepatitis (12/74, 16%). Twenty-nine patients (39%) lived for at least one year, and 16 are still alive one to nine years after transplantation. Technical surgical problems, rejection, and infection were the main causes of death. Improved immunosuppression is needed; nevertheless, the quality of life in the long-term survivors has encouraged continuation of this difficult work. PMID:377201

  19. In Defense of Artificial Replacement.

    PubMed

    Shiller, Derek

    2017-02-03

    If it is within our power to provide a significantly better world for future generations at a comparatively small cost to ourselves, we have a strong moral reason to do so. One way of providing a significantly better world may involve replacing our species with something better. It is plausible that in the not-too-distant future, we will be able to create artificially intelligent creatures with whatever physical and psychological traits we choose. Granted this assumption, it is argued that we should engineer our extinction so that our planet's resources can be devoted to making artificial creatures with better lives.

  20. [Proximal and total femur replacement].

    PubMed

    Pennekamp, P H; Wirtz, D C; Dürr, H R

    2012-07-01

    Reconstruction of segmental bone defects of the proximal femur following wide tumor resection or revision arthroplasty. Aggressive benign or primary malignant bone tumors of the proximal femur; destructive metastases; massive segmental bone defects of the proximal femur; periprosthetic fractures. Local infection; very short life expectancy (< 3 months); massive deficiency of acetabular bone stock. Anterolateral approach. Exposure and detachment of the iliopsoas and gluteus medius muscle from the proximal femur with a sufficient safety margin to the bone; distal transsection of the vastus lateralis/intermedius and rectus femoris muscle according to the extraosseous tumor extension; distal femur osteotomy al least 3 cm beyond the farthest point of tumor extension; in case of total femur replacement, additional lateral arthrotomy of the knee with resection of the ligaments and menisci; reaming of the medullary canal after securing the shaft with a Verbrugge clamp; trial assembly and reduction followed by the definitive implantation of the prosthesis with adjustment of the femoral neck anteversion in 5° increments; soft tissue reconstruction and fixation to an attachment tube covering the prosthesis; in case of total femur replacement, the preparation of the tibia is followed by the coupling of the tibial and femoral components. Infection prophylaxis, 20 kg partial weight bearing, continuous passive motion. A total of 20  patients with proximal femur replacement and 2 patients with total femur replacement implanted between June 2007 and December 2011 were retrospectively reviewed. Three patients had primary malignant bone tumors, while 19 patients underwent resection for metastatic disease. The mean age at surgery was 62.0 ± 18.1 years (18-82 years). Fifteen patients with a mean follow-up of 20.3 ± 17.2 months (4-51 months) were studied. Among the 22 cases, periprosthetic infection occurred in 3 patients (13.6%), dislocation in 2 patients (9

  1. SINE retrotransposons cause epigenetic reprogramming of adjacent gene promoters.

    PubMed

    Estécio, Marcos R H; Gallegos, Juan; Dekmezian, Mhair; Lu, Yue; Liang, Shoudan; Issa, Jean-Pierre J

    2012-10-01

    Almost half of the human genome and as much as 40% of the mouse genome is composed of repetitive DNA sequences. The majority of these repeats are retrotransposons of the SINE and LINE families, and such repeats are generally repressed by epigenetic mechanisms. It has been proposed that these elements can act as methylation centers from which DNA methylation spreads into gene promoters in cancer. Contradictory to a methylation center function, we have found that retrotransposons are enriched near promoter CpG islands that stay methylation-free in cancer. Clearly, it is important to determine which influence, if any, these repetitive elements have on nearby gene promoters. Using an in vitro system, we confirm here that SINE B1 elements can influence the activity of downstream gene promoters, with acquisition of DNA methylation and loss of activating histone marks, thus resulting in a repressed state. SINE sequences themselves did not immediately acquire DNA methylation but were marked by H3K9me2 and H3K27me3. Moreover, our bisulfite sequencing data did not support that gain of DNA methylation in gene promoters occurred by methylation spreading from SINE B1 repeats. Genome-wide analysis of SINE repeats distribution showed that their enrichment is directly correlated with the presence of USF1, USF2, and CTCF binding, proteins with insulator function. In summary, our work supports the concept that SINE repeats interfere negatively with gene expression and that their presence near gene promoters is counter-selected, except when the promoter is protected by an insulator element.

  2. Smokers Prone to Problems After Joint Replacement

    MedlinePlus

    ... fullstory_164137.html Smokers Prone to Problems After Joint Replacement: Study Cessation programs help reduce the rate ... that smokers do worse than non-smokers after joint replacements, and now this research shows there's good ...

  3. Hip Replacement: MedlinePlus Health Topic

    MedlinePlus

    ... Arthritis and Musculoskeletal and Skin Diseases) Also in Spanish Total Hip Replacement (American Academy of Orthopaedic Surgeons) - PDF Also in Spanish Total Hip Replacement (Arthroplasty) (Beyond the Basics) (UpToDate) ...

  4. Knee Replacement: MedlinePlus Health Topic

    MedlinePlus

    ... Arthritis and Musculoskeletal and Skin Diseases) Also in Spanish Knee Replacement (Mayo Foundation for Medical Education and ... American Academy of Orthopaedic Surgeons) - PDF Also in Spanish Total Knee Replacement (Arthroplasty) (Beyond the Basics) (UpToDate) ...

  5. Development of an anhydrotetracycline-inducible gene expression system for solvent-producing Clostridium acetobutylicum: A useful tool for strain engineering.

    PubMed

    Dong, Hongjun; Tao, Wenwen; Zhang, Yanping; Li, Yin

    2012-01-01

    Clostridium acetobutylicum is an important solvent (acetone-butanol-ethanol) producing bacterium. However, a stringent, effective, and convenient-to-use inducible gene expression system that can be used for regulating the gene expression strength in C. acetobutylicum is currently not available. Here, we report an anhydrotetracycline-inducible gene expression system for solvent-producing bacterium C. acetobutylicum. This system consists of a functional chloramphenicol acetyltransferase gene promoter containing tet operators (tetO), Pthl promoter (thiolase gene promoter from C. acetobutylicum) controlling TetR repressor expression cassette, and the chemical inducer anhydrotetracycline (aTc). The optimized system, designated as pGusA2-2tetO1, allows gene regulation in an inducer aTc concentration-dependent way, with an inducibility of over two orders of magnitude. The stringency of TetR repression supports the introduction of the genes encoding counterselective marker into C. acetobutylicum, which can be used to increase the mutant screening efficiency. This aTc-inducible gene expression system will thus increase the genetic manipulation capability for engineering C. acetobutylicum. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. B Plant process piping replacement feasibility study

    SciTech Connect

    Howden, G.F.

    1996-02-07

    Reports on the feasibility of replacing existing embedded process piping with new more corrosion resistant piping between cells and between cells and a hot pipe trench of a Hanford Site style canyon facility. Provides concepts for replacement piping installation, and use of robotics to replace the use of the canyon crane as the primary means of performing/supporting facility modifications (eg, cell lining, pipe replacement, equipment reinstallation) and operational maintenenace.

  7. Total hip replacement in dancers.

    PubMed

    Buyls, Inge R A E; Rietveld, A B M Boni; Ourila, Tiia; Emerton, Mark E; Bird, H A

    2013-04-01

    A case report of a professional contemporary dancer who successfully returned to the stage after bilateral total hip replacements (THR) for osteoarthritis is presented, together with her own commentary and a retrospective cohort study of total hip replacements in dancers. In the presented cohort, there were no post-operative dislocations or infections, the original pain had been relieved, rehabilitation was objectively normal and all resumed their dance (teaching) activities. Nevertheless, they were disappointed about the prolonged rehabilitation. Due to their high demands as professional dancers, post-operative expectations were too optimistic in view of the usual quick and favourable results of THR in the older and less physically active, general population. In all dancers with unilateral osteoarthritis, the left hip was involved, which may reflect the tendency to use the left leg as standing leg and be suggestive that strenuous physical activity may lead to osteoarthritis. Better rehabilitation guidelines are needed for dancer patients undergoing THR, especially drawing their attention to realistic post-operative expectations.

  8. The Mitochondrial Replacement 'Therapy' Myth.

    PubMed

    Rulli, Tina

    2016-12-30

    This article argues that two forms of mitochondrial replacement therapy, maternal spindle transfer (MST) and pro-nuclear transfer (PNT), are not therapies at all because they do not treat children who are coming into existence. Rather, these technologies merely create healthy children where none was inevitable. Even if creating healthy lives has some value, it is not to be confused with the medical value of a cure or therapy. The article addresses a recent Bioethics article, 'Mitochondrial Replacement: Ethics and Identity,' by Wrigley, Wilkinson, and Appleby, who argue that PNT is morally favorable to MST due to the Non-Identity Problem. Wrigley et al. claim that PNT, since it occurs post-conception, preserves the identity of the resulting child, whereas MST, since it occurs pre-conception, is an identity-altering technique. As such, a child born with mitochondrial disease could complain that her parents failed to use PNT, but not MST. The present article argues that the authors are mistaken: both MST and PNT are identity-affecting techniques. But this is of little matter, for we should be cautious in drawing any moral conclusions from the application of the Non-Identity Problem to cases. The article then argues that the authors are mistaken in inferring that PNT is a type of embryonic cure or therapy for children with mitochondrial disease. The article cautions against the mistaken life-saving rhetoric that is common in bioethics discussions of MRTs.

  9. Fetal tissue engineering: diaphragmatic replacement.

    PubMed

    Fauza, D O; Marler, J J; Koka, R; Forse, R A; Mayer, J E; Vacanti, J P

    2001-01-01

    Prosthetic repair of congenital diaphragmatic hernia has been associated with high complication rates. This study was aimed at applying fetal tissue engineering to diaphragmatic replacement. Fetal lambs underwent harvest of skeletal muscle specimens. Once expanded in vitro, fetal myoblasts were suspended in a collagen hydrogel submitted to controlled radial tension. The construct was then placed in a bioreactor. After birth, all animals underwent creation of 2 diaphragmatic defects. One defect was repaired with the autologous-engineered construct placed in between 2 acellular supporting membranes and the other with an identical construct but without any cells. Each animal was its own control (graft, n = 10). Animals were killed at different time-points postimplantation for histologic examination. Statistical analysis was by analysis of variance (ANOVA). Fetal myoblasts expanded up to twice as fast as neonatal cells. Hydrogel-based radial tension enhanced construct architecture by eliciting cell organization within the scaffold. No eventration was present in 4 of 5 engineered constructs but in 0 of 5 acellular grafts (P<.05). At harvest, engineered constructs were thick and histologically resembled normal skeletal muscle, whereas acellular grafts were thin, floppy, and showed low cell density with increased fibrosis. Unlike acellular grafts, engineered cellular diaphragmatic constructs are anatomically and histologically similar to normal muscle. Fetal tissue engineering may be a viable alternative for diaphragmatic replacement.

  10. Should the patella be replaced in total knee replacement?

    PubMed

    Badhe, N; Dewnany, G; Livesley, P J

    2001-01-01

    In 170 total knee arthroplasties for osteoarthritis 71 did not receive a patellar replacement (group A), while 99 knees had a cemented polyethylene patella (group B). The mean follow-up time was 36 months (30-50 months). In group A 10 patients underwent second-stage patellar resurfacing and in group B 2 knees underwent revision of the patellar component. Radiologically the average patellar congruency was similar. In both groups there were 21 non-congruent knees. In group A 8 were symptomatic and had low scores compared to 2 in group B (P<0.05). The mean HSS score and patellar score were higher in group B than in group A (P<0.05).

  11. Hip Replacement - Multiple Languages: MedlinePlus

    MedlinePlus

    ... Hip Replacement (Arabic) العربية Bilingual PDF Health Information Translations Bosnian (Bosanski) Total Hip Replacement Potpuna zamjena kuka - Bosanski (Bosnian) Bilingual PDF Health Information Translations Chinese - Simplified (简体中文) Total Hip Replacement 全髋关节置换 - 简体中文 ( ...

  12. Knee Replacement - Multiple Languages: MedlinePlus

    MedlinePlus

    ... Knee Replacement (Arabic) العربية Bilingual PDF Health Information Translations Bosnian (Bosanski) Total Knee Replacement Potpuna zamjena koljena - Bosanski (Bosnian) Bilingual PDF Health Information Translations Chinese - Simplified (简体中文) Total Knee Replacement 全膝关节置换 - 简体中文 ( ...

  13. 22 CFR 51.10 - Replacement passports.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Replacement passports. 51.10 Section 51.10 Foreign Relations DEPARTMENT OF STATE NATIONALITY AND PASSPORTS PASSPORTS General § 51.10 Replacement passports. A passport issuing office may issue a replacement passport without payment of applicable fees for...

  14. 22 CFR 51.10 - Replacement passports.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 22 Foreign Relations 1 2013-04-01 2013-04-01 false Replacement passports. 51.10 Section 51.10 Foreign Relations DEPARTMENT OF STATE NATIONALITY AND PASSPORTS PASSPORTS General § 51.10 Replacement passports. A passport issuing office may issue a replacement passport without payment of applicable fees for...

  15. 22 CFR 51.10 - Replacement passports.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 22 Foreign Relations 1 2011-04-01 2011-04-01 false Replacement passports. 51.10 Section 51.10 Foreign Relations DEPARTMENT OF STATE NATIONALITY AND PASSPORTS PASSPORTS General § 51.10 Replacement passports. A passport issuing office may issue a replacement passport without payment of applicable fees for...

  16. 22 CFR 51.10 - Replacement passports.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 22 Foreign Relations 1 2012-04-01 2012-04-01 false Replacement passports. 51.10 Section 51.10 Foreign Relations DEPARTMENT OF STATE NATIONALITY AND PASSPORTS PASSPORTS General § 51.10 Replacement passports. A passport issuing office may issue a replacement passport without payment of applicable fees for...

  17. 22 CFR 51.10 - Replacement passports.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 22 Foreign Relations 1 2014-04-01 2014-04-01 false Replacement passports. 51.10 Section 51.10 Foreign Relations DEPARTMENT OF STATE NATIONALITY AND PASSPORTS PASSPORTS General § 51.10 Replacement passports. A passport issuing office may issue a replacement passport without payment of applicable fees for...

  18. Molecular replacement then and now

    SciTech Connect

    Scapin, Giovanna

    2013-11-01

    A brief overview, with examples, of the evolution of molecular-replacement methods and models over the past few years is presented. The ‘phase problem’ in crystallography results from the inability to directly measure the phases of individual diffracted X-ray waves. While intensities are directly measured during data collection, phases must be obtained by other means. Several phasing methods are available (MIR, SAR, MAD, SAD and MR) and they all rely on the premise that phase information can be obtained if the positions of marker atoms in the unknown crystal structure are known. This paper is dedicated to the most popular phasing method, molecular replacement (MR), and represents a personal overview of the development, use and requirements of the methodology. The first description of noncrystallographic symmetry as a tool for structure determination was explained by Rossmann and Blow [Rossmann & Blow (1962 ▶), Acta Cryst.15, 24–31]. The term ‘molecular replacement’ was introduced as the name of a book in which the early papers were collected and briefly reviewed [Rossmann (1972 ▶), The Molecular Replacement Method. New York: Gordon & Breach]. Several programs have evolved from the original concept to allow faster and more sophisticated searches, including six-dimensional searches and brute-force approaches. While careful selection of the resolution range for the search and the quality of the data will greatly influence the outcome, the correct choice of the search model is probably still the main criterion to guarantee success in solving a structure using MR. Two of the main parameters used to define the ‘best’ search model are sequence identity (25% or more) and structural similarity. Another parameter that may often be undervalued is the quality of the probe: there is clearly a relationship between the quality and the correctness of the chosen probe and its usefulness as a search model. Efforts should be made by all structural biologists to

  19. Cell and gene therapy.

    PubMed

    Rao, Rajesh C; Zacks, David N

    2014-01-01

    Replacement or repair of a dysfunctional gene combined with promoting cell survival is a two-pronged approach that addresses an unmet need in the therapy of retinal degenerative diseases. In this chapter, we discuss various strategies toward achieving both goals: transplantation of wild-type cells to replace degenerating cells and to rescue gene function, sequential gene and cell therapy, and in vivo reprogramming of rods to cones. These approaches highlight cutting-edge advances in cell and gene therapy, and cellular lineage conversion in order to devise new therapies for various retinal degenerative diseases.

  20. Mitochondrial Replacement: Ethics and Identity.

    PubMed

    Wrigley, Anthony; Wilkinson, Stephen; Appleby, John B

    2015-11-01

    Mitochondrial replacement techniques (MRTs) have the potential to allow prospective parents who are at risk of passing on debilitating or even life-threatening mitochondrial disorders to have healthy children to whom they are genetically related. Ethical concerns have however been raised about these techniques. This article focuses on one aspect of the ethical debate, the question of whether there is any moral difference between the two types of MRT proposed: Pronuclear Transfer (PNT) and Maternal Spindle Transfer (MST). It examines how questions of identity impact on the ethical evaluation of each technique and argues that there is an important difference between the two. PNT, it is argued, is a form of therapy based on embryo modification while MST is, instead, an instance of selective reproduction. The article's main ethical conclusion is that, in some circumstances, there is a stronger obligation to use PNT than MST.

  1. Replacement Sequence of Events Generator

    NASA Technical Reports Server (NTRS)

    Fisher, Forest; Gladden, Daniel Wenkert Roy; Khanampompan, Teerpat

    2008-01-01

    The soeWINDOW program automates the generation of an ITAR (International Traffic in Arms Regulations)-compliant sub-RSOE (Replacement Sequence of Events) by extracting a specified temporal window from an RSOE while maintaining page header information. RSOEs contain a significant amount of information that is not ITAR-compliant, yet that foreign partners need to see for command details to their instrument, as well as the surrounding commands that provide context for validation. soeWINDOW can serve as an example of how command support products can be made ITAR-compliant for future missions. This software is a Perl script intended for use in the mission operations UNIX environment. It is designed for use to support the MRO (Mars Reconnaissance Orbiter) instrument team. The tool also provides automated DOM (Distributed Object Manager) storage into the special ITAR-okay DOM collection, and can be used for creating focused RSOEs for product review by any of the MRO teams.

  2. Optimal randomized scheduling by replacement

    SciTech Connect

    Saias, I.

    1996-05-01

    In the replacement scheduling problem, a system is composed of n processors drawn from a pool of p. The processors can become faulty while in operation and faulty processors never recover. A report is issued whenever a fault occurs. This report states only the existence of a fault but does not indicate its location. Based on this report, the scheduler can reconfigure the system and choose another set of n processors. The system operates satisfactorily as long as, upon report of a fault, the scheduler chooses n non-faulty processors. We provide a randomized protocol maximizing the expected number of faults the system can sustain before the occurrence of a crash. The optimality of the protocol is established by considering a closely related dual optimization problem. The game-theoretic technical difficulties that we solve in this paper are very general and encountered whenever proving the optimality of a randomized algorithm in parallel and distributed computation.

  3. Mitochondrial Replacement: Ethics and Identity

    PubMed Central

    Wilkinson, Stephen; Appleby, John B.

    2015-01-01

    Abstract Mitochondrial replacement techniques (MRTs) have the potential to allow prospective parents who are at risk of passing on debilitating or even life‐threatening mitochondrial disorders to have healthy children to whom they are genetically related. Ethical concerns have however been raised about these techniques. This article focuses on one aspect of the ethical debate, the question of whether there is any moral difference between the two types of MRT proposed: Pronuclear Transfer (PNT) and Maternal Spindle Transfer (MST). It examines how questions of identity impact on the ethical evaluation of each technique and argues that there is an important difference between the two. PNT, it is argued, is a form of therapy based on embryo modification while MST is, instead, an instance of selective reproduction. The article's main ethical conclusion is that, in some circumstances, there is a stronger obligation to use PNT than MST. PMID:26481204

  4. ORO. The physical developer replacement?

    PubMed

    Wood, Michael A; James, Tim

    2009-12-01

    In the process of fingerprint development Physical Developer has been largely the method of choice on porous surfaces after coming into contact with wet environments. It is only recently that a new technique has been identified which could replace this standard technique. This study aims to build on previous research and expand knowledge regarding the technique. The study built on previous research and compared Physical Developer to Oil Red O, testing both on four paper types, while being placed in three different water types and an accelerant for various amounts of time. Marks were placed with both heavily 'loaded' sebaceous fingers and 'normal' un-washed fingers. Results show that Oil Red O consistently produced clearer more detailed marks from the 'loaded' fingers, but neither technique proved to work better on the 'normal' marks. Neither technique developed any prints from the accelerant.

  5. Perspectives in hormone replacement therapy.

    PubMed

    Kenemans, P; van Unnik, G A; Mijatovic, V; van der Mooren, M J

    2001-06-15

    Estrogens have been convincingly shown to be highly effective in preventing and reversing menopause-related conditions, such as hot flushes, urogenital complaints, and postmenopausal bone loss. Observational studies report that long-term, estrogen-containing, postmenopausal hormone replacement therapy (HRT) leads to a substantial reduction in hip fractures, myocardial infarction, and possibly colonic cancer, with important consequences for health and quality of life. Estrogen replacement may postpone the onset of Alzheimer's disease and extend life. While many of these effects are biologically plausible, with a variety of cellular mechanisms being involved, only ongoing and future large-scale randomized clinical trials can and should define the effects of HRT more precisely. Long-term compliance is a key issue for long-term benefits, and offering women a choice of administration routes and regimens can only be beneficial in this respect. Pills, patches, gels, and implants are all widely prescribed. Intravaginal or intranasal forms of administration, which are very easy to use and adaptable on an individual level, are among the new options which could improve long-term continuation of HRT use. Fear of breast cancer and recurrence of vaginal bleeding are real concerns for many women considering HRT. This has led to research into lower-dose, estrogen-containing regimens, into continuous combined regimens, and into the potential of estrogen receptor alpha or beta binding molecules that may help to prevent such problems from arising. The prospects for safe and effective postmenopausal HRT with either estrogens or estrogen-like drugs are very promising when these drugs are used in a patient-tailored, risk profile-based manner.

  6. Dental ontogeny and replacement in Pliosauridae.

    PubMed

    Sassoon, Judyth; Foffa, Davide; Marek, Ryan

    2015-11-01

    Dental morphology and patterns of tooth replacement in representatives of the clade Pliosauridae (Reptilia, Sauropterygia) are evaluated in detail. The jaws of one basal (Thalassiodracon hawkinsii) and two derived species (Pliosaurus carpenteri, Pliosaurus kevani) were visualized by μCT scans, and the ontogenetic patterns, or 'movement paths', of replacement teeth could be mapped. Other specimens (Peloneustes philarchus and Pliosaurus westbuyensis) with well-preserved jaws containing functional and replacement teeth in situ were also examined directly, and waves of tooth replacement could be inferred from the degree of in situ tooth development and the fusion between functional and replacement alveoli. The analysis revealed symmetrical tooth eruption over the medial axis throughout the length of the jaw in the basal pliosaurid Thalassiodracon. By contrast, symmetrical tooth eruption patterns occur only along the anterior sections of the jaws of derived pliosaurids. In Pliosaurus, replacement schedules differ in the anterior and posterior portions of the jaws and appear to correlate with differences in tooth morphology and symmetrical replacement. The anterior teeth exhibit longer replacement cycle periods and symmetrical replacement, while shorter cycle periods and asymmetry are seen posteriorly. A longer period suggests slower replacement and is characteristic of large, specialized caniniform teeth in the longer snouted Late Jurassic taxa. Smaller posterior teeth have a shorter period and therefore a faster replacement cycle. The transition from long to short replacement period over the length of the jaw is thought to account for the loss of symmetry. This differentiation could relate to differential tooth function and a type of heterodonty. We therefore propose a new model of pliosaurid tooth replacement patterns and present it in a phylogenetic context.

  7. Dental ontogeny and replacement in Pliosauridae

    PubMed Central

    Sassoon, Judyth; Foffa, Davide; Marek, Ryan

    2015-01-01

    Dental morphology and patterns of tooth replacement in representatives of the clade Pliosauridae (Reptilia, Sauropterygia) are evaluated in detail. The jaws of one basal (Thalassiodracon hawkinsii) and two derived species (Pliosaurus carpenteri, Pliosaurus kevani) were visualized by μCT scans, and the ontogenetic patterns, or ‘movement paths’, of replacement teeth could be mapped. Other specimens (Peloneustes philarchus and Pliosaurus westbuyensis) with well-preserved jaws containing functional and replacement teeth in situ were also examined directly, and waves of tooth replacement could be inferred from the degree of in situ tooth development and the fusion between functional and replacement alveoli. The analysis revealed symmetrical tooth eruption over the medial axis throughout the length of the jaw in the basal pliosaurid Thalassiodracon. By contrast, symmetrical tooth eruption patterns occur only along the anterior sections of the jaws of derived pliosaurids. In Pliosaurus, replacement schedules differ in the anterior and posterior portions of the jaws and appear to correlate with differences in tooth morphology and symmetrical replacement. The anterior teeth exhibit longer replacement cycle periods and symmetrical replacement, while shorter cycle periods and asymmetry are seen posteriorly. A longer period suggests slower replacement and is characteristic of large, specialized caniniform teeth in the longer snouted Late Jurassic taxa. Smaller posterior teeth have a shorter period and therefore a faster replacement cycle. The transition from long to short replacement period over the length of the jaw is thought to account for the loss of symmetry. This differentiation could relate to differential tooth function and a type of heterodonty. We therefore propose a new model of pliosaurid tooth replacement patterns and present it in a phylogenetic context. PMID:26715998

  8. [Animal experiment, can we replace?

    PubMed

    Combrisson, H

    2017-09-01

    Animal experiment is a subject of controversies. Some people, defenders of animals, think that it is not acceptable to use for scientific purposes at the risk of making them suffer or assert that the results obtained with animals are not transposable in the human beings. Others, in particular researchers in biology or medicine, think that the animal models are essential for the biomedical search. This confrontation of the opinions bases largely on an evolution of the place of animals in our society. The regulations authorize the use of animals for scientific purposes but oblige to make it under restrictive conditions. The application of 3Rs - replacement, reduction, and refinement - expressed in 1959 by Russel and Burch is an ethical guide to improve the welfare of animals in research. The alternative methods do not allow, in the present state of the knowledge, to answer all the scientific questions in biology and medicine research. They are, most of the time, complementary methods of the in vivo methods. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  9. Visual Image Sensor Organ Replacement

    NASA Technical Reports Server (NTRS)

    Maluf, David A.

    2014-01-01

    This innovation is a system that augments human vision through a technique called "Sensing Super-position" using a Visual Instrument Sensory Organ Replacement (VISOR) device. The VISOR device translates visual and other sensors (i.e., thermal) into sounds to enable very difficult sensing tasks. Three-dimensional spatial brightness and multi-spectral maps of a sensed image are processed using real-time image processing techniques (e.g. histogram normalization) and transformed into a two-dimensional map of an audio signal as a function of frequency and time. Because the human hearing system is capable of learning to process and interpret extremely complicated and rapidly changing auditory patterns, the translation of images into sounds reduces the risk of accidentally filtering out important clues. The VISOR device was developed to augment the current state-of-the-art head-mounted (helmet) display systems. It provides the ability to sense beyond the human visible light range, to increase human sensing resolution, to use wider angle visual perception, and to improve the ability to sense distances. It also allows compensation for movement by the human or changes in the scene being viewed.

  10. Engineering Alloplastic Temporomandibular Joint Replacements

    PubMed Central

    Sinno, Hani; Tahiri, Youssef; Gilardino, Mirko; Bobyn, Dennis

    2011-01-01

    Temporomandibular disorders (TMD) are part of a heterogeneous group of pathologies that manifest with a constellation of signs and symptoms. They are the most frequent cause of chronic orofacial pain and are prevalent in 12% of the general population. Despite the debilitating nature of these disorders, there is no standardization for treatment of the diseased temporomandibular joint (TMJ). In this review, we present an overview of the functional anatomy of the TMJ and the engineering concepts that must be understood to better understand the indications for surgical management, the types of available treatments and the requirements for reconstruction. A comparison is made of the clinical outcomes with autogenous versus alloplastic reconstruction, including a history of alloplastic materials and the design features of currently available implants. Emphasis is made on material selection, modulus, stiffness, notch sensitivity and modularity. For the treatment of TMD, engineered TMJ alloplastic replacements have had considerable promise with additional room for improvement using new materials and recent design concepts. PMID:22363183

  11. Establishing a Markerless Genetic Exchange System for Methanosarcina mazei Strain Gö1 for Constructing Chromosomal Mutants of Small RNA Genes

    PubMed Central

    Ehlers, Claudia; Jäger, Dominik; Schmitz, Ruth A.

    2011-01-01

    A markerless genetic exchange system was successfully established in Methanosarcina mazei strain Gö1 using the hpt gene coding for hypoxanthine phosphoribosyltransferase. First, a chromosomal deletion mutant of the hpt gene was generated conferring resistance to the purine analog 8-aza-2,6-diaminopurine (8-ADP). The nonreplicating allelic exchange vector (pRS345) carrying the pac-resistance cassette for direct selection of chromosomal integration, and the hpt gene for counterselection was introduced into this strain. By a pop-in and ultimately pop-out event of the plasmid from the chromosome, allelic exchange is enabled. Using this system, we successfully generated a M. mazei deletion mutant of the gene encoding the regulatory non-coding RNA sRNA154. Characterizing M. mazeiΔsRNA 154 under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA154 in regulation of nitrogen fixation by posttranscriptional regulation. PMID:21941461

  12. Biology of tooth replacement in amniotes

    PubMed Central

    Whitlock, John A; Richman, Joy M

    2013-01-01

    Tooth replacement is a common trait to most vertebrates, including mammals. Mammals, however, have lost the capacity for continuous tooth renewal seen in most other vertebrates, and typically have only 1–2 generations of teeth. Here, we review the mechanisms of tooth replacement in reptiles and mammals, and discuss in detail the current and historical theories on control of timing and pattern of tooth replacement and development. PMID:23788284

  13. Arthroscopic treatment of unstable total hip replacement.

    PubMed

    Cuéllar, Ricardo; Aguinaga, Iñaki; Corcuera, Irene; Ponte, Juan; Usabiaga, Jaime

    2010-06-01

    Hip arthroscopy may be useful in the diagnosis and treatment of apparently well-implanted but unstable total hip replacement prostheses. We present 2 cases of arthroscopically assisted capsular tightening in unstable total hip replacements. Both cases had significant capsular laxity. Case 2 had impingement of the lower part of the acetabulum with the lesser trochanter that caused hip dislocation. Early revision surgery can be avoided with the use of this technique in selected cases of unstable total hip replacements.

  14. Environmentally Assisted Cracking: Concerns for Cadmium Replacement

    DTIC Science & Technology

    2003-12-01

    Environmentally Assisted Cracking Concerns for Cadmium Replacement by Scott M. Grendahl ARL-TR-3099 December 2003...5069 ARL-TR-3099 December 2003 Environmentally Assisted Cracking: Concerns for Cadmium Replacement Scott M. Grendahl Weapons and...Concerns for Cadmium Replacement 5c. PROGRAM ELEMENT NUMBER 5d. PROJECT NUMBER 181E31 5e. TASK NUMBER 6. AUTHOR(S) Grendahl, S.M. (ARL) 5f

  15. Total quality management approach improves medication replacement.

    PubMed

    Anderson, L K

    1994-07-01

    Total quality management (TQM) is based on understanding customer needs, improving key processes that affect customer satisfaction, and creating cross-functional teams to resolve process problems. This article describes application of TQM principles and problem-solving processes to improve one OR's medication exchange system. The problem was excessive monthly pharmacy medication replacement costs. The goal was to reduce the monthly medication replacement costs by 50%. Within four months, monthly medication replacement charges decreased from $656 to $302, and by one year, monthly charges decreased to $160. The new process had fewer steps, fewer staff members involved, and fewer delays in medication replacement.

  16. Opportunistic replacement of fusion power system parts

    SciTech Connect

    Day, J.A.; George, L.L.

    1981-10-26

    This paper describes a maintenance problem in a fusion power plant. The problem is to specify which life limited parts should be replaced when there is an opportunity. The objective is to minimize the cost rate of replacement parts and of maintenance actions while satisfying a power plant availability constraint. The maintenance policy is to look ahead and replace all parts that will reach their life limits within a time called a screen. Longer screens yield greater system availabilities because more parts are replaced prior to their life limits.

  17. Peach bottom recirculation piping replacement ALARA program

    SciTech Connect

    Englesson, G.A.; Hilsmeier, A.E.; Mann, B.J.

    1986-01-01

    In late 1983, Philadelphia Electric Company (PECo) began detailed planning to replace the recirculation, residual heat removal, and part of the reactor water cleanup piping of the Peach Bottom Unit 2 reactor. Included in this work was an estimate of the collective exposure expected during piping replacement. That initial estimate, 1945 man-rem, is compared with the actual collective dose incurred during the piping replacement program. Also included are the exposures incurred during two additional tasks (safe end replacement and recirculation pump disassembly and decontamination) not considered in the initial estimate.

  18. Weigh and wait: the prospect of mitochondrial gene replacement.

    PubMed

    Xu, Liang; Shi, Rui

    2016-12-01

    Mitochondrial DNA transfer has recently received attention from physicians. The transfer techniques place genetic material from the egg nucleus of a woman with a mitochondrial DNA mutation into a healthy donated egg from which the nuclear DNA was removed. This technology intends to reconstruct a mitochondria-competent egg to produce a baby. Three approaches: (1) pronuclear transfer; (2) metaphase II spindle transfer (ST); and (3) polar body (PB) transfer, have been proposed and applied in animal models with very low levels of heteroplasmy. Because there is no curative treatment for patients with mitochondrial dysfunction, the UK government has allowed the use of this pioneering technique to prevent the transmission of rare and devastating mitochondrial diseases. Despite general safety in the observation period, this technology involves germline modification, raising scientific and ethical questions in the public. In this review, we focus on this unprecedented technology and discuss its clinical application in the future.

  19. Biosynthetic Pathways, Gene Replacement and the Antiquity of Life

    NASA Astrophysics Data System (ADS)

    Blankenship, R.; Raymond, J.

    2004-12-01

    Our understanding of the evolution and diversity of life during the Archean has been profoundly influenced by geological signatures including microfossils, stromatolites, and fractionated carbon and sulfur isotopes. These biosignatures have been extensively debated on the grounds of competing abiotic processes. Hopane and squalene biomarkers have been taken as evidence that by 2700 Ma, all three domains of life had emerged, that cyanobacteria, the `inventors' of oxygenic photosynthesis, had diverged from their bacterial ancestors, heralding the impending oxidation of Earth's atmosphere, and that O2 was already available, as it is required explicitly in modern organisms for the synthesis of squalene. We argue that, based on corollaries present throughout enzymology, the oxidization of squalene during the Archean could just as likely have been carried out by an anaerobic enzyme that either remains to be characterized or that may have been totally lost as eukaryotes assumed primarily aerobic lifestyles. The transition from an anoxic to an oxic world had unfathomable consequences for early life; molecular oxygen and its derivatives went from being poisonous in an obligately anaerobic world, to assuming a central role in the subsequent development of complex, macroscopic life. Though the aftermath was global in scale, the biological transition to aerobiosis was effected at the molecular level, which included the evolution of a wealth of new enzymes, the extensive modification of many existing enzymes, and the `rewiring' of central biochemical pathways.

  20. Quantifying homologous replacement of loci between haloarchaeal species.

    PubMed

    Williams, David; Gogarten, J Peter; Papke, R Thane

    2012-01-01

    In vitro studies of the haloarchaeal genus Haloferax have demonstrated their ability to frequently exchange DNA between species, whereas rates of homologous recombination estimated from natural populations in the genus Halorubrum are high enough to maintain random association of alleles between five loci. To quantify the effects of gene transfer and recombination of commonly held (relaxed core) genes during the evolution of the class Halobacteria (haloarchaea), we reconstructed the history of 21 genomes representing all major groups. Using a novel algorithm and a concatenated ribosomal protein phylogeny as a reference, we created a directed horizontal genetic transfer (HGT) network of contemporary and ancestral genomes. Gene order analysis revealed that 90% of testable HGTs were by direct homologous replacement, rather than nonhomologous integration followed by a loss. Network analysis revealed an inverse log-linear relationship between HGT frequency and ribosomal protein evolutionary distance that is maintained across the deepest divergences in Halobacteria. We use this mathematical relationship to estimate the total transfers and amino acid substitutions delivered by HGTs in each genome, providing a measure of chimerism. For the relaxed core genes of each genome, we conservatively estimate that 11-20% of their evolution occurred in other haloarchaea. Our findings are unexpected, because the transfer and homologous recombination of relaxed core genes between members of the class Halobacteria disrupts the coevolution of genes; however, the generation of new combinations of divergent but functionally related genes may lead to adaptive phenotypes not available through cumulative mutations and recombination within a single population.

  1. Quantifying Homologous Replacement of Loci between Haloarchaeal Species

    PubMed Central

    Williams, David; Gogarten, J. Peter; Papke, R. Thane

    2012-01-01

    In vitro studies of the haloarchaeal genus Haloferax have demonstrated their ability to frequently exchange DNA between species, whereas rates of homologous recombination estimated from natural populations in the genus Halorubrum are high enough to maintain random association of alleles between five loci. To quantify the effects of gene transfer and recombination of commonly held (relaxed core) genes during the evolution of the class Halobacteria (haloarchaea), we reconstructed the history of 21 genomes representing all major groups. Using a novel algorithm and a concatenated ribosomal protein phylogeny as a reference, we created a directed horizontal genetic transfer (HGT) network of contemporary and ancestral genomes. Gene order analysis revealed that 90% of testable HGTs were by direct homologous replacement, rather than nonhomologous integration followed by a loss. Network analysis revealed an inverse log-linear relationship between HGT frequency and ribosomal protein evolutionary distance that is maintained across the deepest divergences in Halobacteria. We use this mathematical relationship to estimate the total transfers and amino acid substitutions delivered by HGTs in each genome, providing a measure of chimerism. For the relaxed core genes of each genome, we conservatively estimate that 11–20% of their evolution occurred in other haloarchaea. Our findings are unexpected, because the transfer and homologous recombination of relaxed core genes between members of the class Halobacteria disrupts the coevolution of genes; however, the generation of new combinations of divergent but functionally related genes may lead to adaptive phenotypes not available through cumulative mutations and recombination within a single population. PMID:23160063

  2. Reoperations Following Cervical Disc Replacement

    PubMed Central

    Skovrlj, Branko; Lee, Dong-Ho; Caridi, John Michael

    2015-01-01

    Cervical disc replacement (CDR) has emerged as an alternative surgical option to cervical arthrodesis. With increasing numbers of patients and longer follow-ups, complications related to the device and/or aging spine are growing, leaving us with a new challenge in the management and surgical revision of CDR. The purpose of this study is to review the current literature regarding reoperations following CDR and to discuss about the approaches and solutions for the current and future potential complications associated with CDR. The published rates of reoperation (mean, 1.0%; range, 0%-3.1%), revision (mean, 0.2%; range, 0%-0.5%), and removal (mean, 1.2%; range, 0%-1.9%) following CDR are low and comparable to the published rates of reoperation (mean, 1.7%; range; 0%-3.4%), revision (mean, 1.5%; range, 0%-4.7%), and removal (mean, 2.0%; range, 0%-3.4%) following cervical arthrodesis. The surgical interventions following CDR range from the repositioning to explantation followed by fusion or the reimplantation to posterior foraminotomy or fusion. Strict patient selection, careful preoperative radiographic review and surgical planning, as well as surgical technique may reduce adverse events and the need for future intervention. Minimal literature and no guidelines exist for the approaches and techniques in revision and for the removal of implants following CDR. Adherence to strict indications and precise surgical technique may reduce the number of reoperations, revisions, and removals following CDR. Long-term follow-up studies are needed, assessing the implant survivorship and its effect on the revision and removal rates. PMID:26097667

  3. Total joint replacement preadmission programs.

    PubMed

    Messer, B

    1998-01-01

    Patients begin to formulate their expectations of the postoperative hospitalization during the preadmission program. The challenge is to better understand the factors patients consider when formulating judgments about the quality of preadmission education. For example, it may be that perceptions of the preadmission program are influenced by what patients believe about their postoperative pain and functional abilities. Specific attention needs to be given both preoperatively and postoperatively to instructing patients on realistic expectations for recovery. One other method of measuring patient outcomes is with the Health Status Profile (SF-36) (Response Healthcare Information Management, 1995). The SF-36 approach emphasizes the outcome of medical care as the patient sees it, in addition to a clinical evaluation of successful health care. This form is currently initiated in the physician's office and returned for scanning at the preadmission class. The patient then completes another SF-36 at 6 months and every year thereafter to compare measurable outcomes. Patients intending to have elective total joint replacements experience anxiety and require much support and education. An effective preadmission program is a major investment in a patient's recovery, as well as a unique marketing tool to customers. Preadmission programs can be viewed as an opportunity to enhance customer satisfaction. Preadmission clinics are an excellent means for nurses to improve the quality of patient care through patient education. the overall goal of preadmission testing programs is to ensure patient preparedness while increasing quality health care and overall customer satisfaction. To enhance program effectiveness, health care providers must lead collaborative efforts to improve the efficiency of systems.

  4. 48 CFR 8.715 - Replacement commodities.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 1 2014-10-01 2014-10-01 false Replacement commodities. 8.715 Section 8.715 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION ACQUISITION... Are Blind or Severely Disabled 8.715 Replacement commodities. When a commodity on the Procurement List...

  5. 48 CFR 8.715 - Replacement commodities.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false Replacement commodities. 8.715 Section 8.715 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION ACQUISITION... Are Blind or Severely Disabled 8.715 Replacement commodities. When a commodity on the Procurement List...

  6. 30 CFR 823.14 - Soil replacement.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Soil replacement. (a) Soil reconstruction specifications established by the U.S. Soil Conservation Service shall be based upon the standards of the National Cooperative Soil Survey and shall include, as a... 30 Mineral Resources 3 2012-07-01 2012-07-01 false Soil replacement. 823.14 Section 823.14 Mineral...

  7. 30 CFR 823.14 - Soil replacement.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Soil replacement. (a) Soil reconstruction specifications established by the U.S. Soil Conservation Service shall be based upon the standards of the National Cooperative Soil Survey and shall include, as a... 30 Mineral Resources 3 2013-07-01 2013-07-01 false Soil replacement. 823.14 Section 823.14 Mineral...

  8. 30 CFR 823.14 - Soil replacement.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Soil replacement. (a) Soil reconstruction specifications established by the U.S. Soil Conservation Service shall be based upon the standards of the National Cooperative Soil Survey and shall include, as a... 30 Mineral Resources 3 2014-07-01 2014-07-01 false Soil replacement. 823.14 Section 823.14 Mineral...

  9. 30 CFR 823.14 - Soil replacement.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Soil replacement. (a) Soil reconstruction specifications established by the U.S. Soil Conservation Service shall be based upon the standards of the National Cooperative Soil Survey and shall include, as a... 30 Mineral Resources 3 2010-07-01 2010-07-01 false Soil replacement. 823.14 Section 823.14 Mineral...

  10. 30 CFR 823.14 - Soil replacement.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Soil replacement. (a) Soil reconstruction specifications established by the U.S. Soil Conservation Service shall be based upon the standards of the National Cooperative Soil Survey and shall include, as a... 30 Mineral Resources 3 2011-07-01 2011-07-01 false Soil replacement. 823.14 Section 823.14 Mineral...

  11. International variation in hip replacement rates

    PubMed Central

    Merx, H; Dreinhofer, K; Schrader, P; Sturmer, T; Puhl, W; Gunther, K; Brenner, H

    2003-01-01

    Objectives: To summarise epidemiological data on the frequency of hip replacements in the countries of the developed world, especially in countries of the Organisation for Economic Cooperation and Development (OECD), and to investigate whether missing consensus criteria for the indication for total hip replacement (THR) result in different replacement rates. Methods: Country-specific hip replacement rates were collected using the available literature, different data sources of national authorities, and estimates of leading hip replacement manufacturers. Results: According to administrative and literature data sources the reported crude primary THR rate varied between 50 and 130 procedures/100 000 inhabitants in OECD countries in the 1990s. The crude overall hip implantation rate, summarising THR, partial hip replacement, and hip revision procedures, was reported to range from 60 to 200 procedures/100 000 inhabitants in the late 1990s. Moreover, large national differences were seen in the relationship between total and partial hip replacement procedures. Conclusion: The reported differences in hip replacement rates in OECD countries are substantial. They may be due to various causes, including different coding systems, country-specific differences in the healthcare system, in total expenditure on health per capita, in the population age structure, and in different indication criteria for THR. PMID:12594106

  12. Mercury Thermometer Replacements in Chemistry Laboratories

    ERIC Educational Resources Information Center

    Foster, Barbara L.

    2005-01-01

    The consequences of broken mercury-in-glass thermometers in academic laboratories results in various health and environmental hazards, which needs to be replaced, by long-stem digital thermometers and non-mercury glass thermometers. The factors that should be considered during the mercury replacement process are types of applications in the…

  13. Mercury Thermometer Replacements in Chemistry Laboratories

    ERIC Educational Resources Information Center

    Foster, Barbara L.

    2005-01-01

    The consequences of broken mercury-in-glass thermometers in academic laboratories results in various health and environmental hazards, which needs to be replaced, by long-stem digital thermometers and non-mercury glass thermometers. The factors that should be considered during the mercury replacement process are types of applications in the…

  14. 7 CFR 1944.659 - Replacement housing.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 13 2010-01-01 2009-01-01 true Replacement housing. 1944.659 Section 1944.659 Agriculture Regulations of the Department of Agriculture (Continued) RURAL HOUSING SERVICE, RURAL BUSINESS...) PROGRAM REGULATIONS (CONTINUED) HOUSING Housing Preservation Grants § 1944.659 Replacement...

  15. 7 CFR 1944.659 - Replacement housing.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 13 2014-01-01 2013-01-01 true Replacement housing. 1944.659 Section 1944.659 Agriculture Regulations of the Department of Agriculture (Continued) RURAL HOUSING SERVICE, RURAL BUSINESS...) PROGRAM REGULATIONS (CONTINUED) HOUSING Housing Preservation Grants § 1944.659 Replacement...

  16. 7 CFR 1944.659 - Replacement housing.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 13 2013-01-01 2013-01-01 false Replacement housing. 1944.659 Section 1944.659 Agriculture Regulations of the Department of Agriculture (Continued) RURAL HOUSING SERVICE, RURAL BUSINESS...) PROGRAM REGULATIONS (CONTINUED) HOUSING Housing Preservation Grants § 1944.659 Replacement...

  17. [Themistocles Gluck. 100 years artificial joint replacement].

    PubMed

    Wessinghage, D

    1991-01-01

    One hundred years ago Themistocles Gluck reported for the first time about artificial joint replacements and the fixation by cement in a most important publication. Extirpation even of internal organs and differentiated possibilities of their replacement were standing in the center of the scientific work of this genius but first of all controversial discussed German doctor.

  18. Applying stand replacement prescribed fires in Alaska

    Treesearch

    Larry A. Vanderlinden

    1996-01-01

    Stand replacement prescribed burning has been applied in Alaska on several occasions. Based on that experience, perspectives can be provided, issues can be discussed, and keys to success can be identified that are applicable to stand replacement prescribed burning activities in areas outside Alaska.

  19. Musings on genome medicine: enzyme-replacement therapy of the lysosomal storage diseases

    PubMed Central

    2009-01-01

    The lysosomal storage diseases, such as Gaucher's disease, mucopolysaccharidosis I, II and IV, Fabry's disease, and Pompe's disease, are rare inherited disorders whose symptoms result from enzyme deficiency causing lysosomal accumulation. Until effective gene-replacement therapy is developed, expensive, and at best incomplete, enzyme-replacement therapy is the only hope for sufferers of rare lysosomal storage diseases. Preventive strategies involving carrier detection should be a priority toward the successful management of these conditions. PMID:20017892

  20. Enzyme replacement therapy for murine hypophosphatasia.

    PubMed

    Millán, José Luis; Narisawa, Sonoko; Lemire, Isabelle; Loisel, Thomas P; Boileau, Guy; Leonard, Pierre; Gramatikova, Svetlana; Terkeltaub, Robert; Camacho, Nancy Pleshko; McKee, Marc D; Crine, Philippe; Whyte, Michael P

    2008-06-01

    Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5'-phosphate (PLP), a co-factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6-dependent seizures. There is no established medical treatment. Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one-step purification and a deca-aspartate sequence (D10) for targeting to mineralizing tissue (sALP-FcD10). TNALP-null mice (Akp2-/-), an excellent model for infantile HPP, were treated from birth using sALP-FcD10. Short-term and long-term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP-FcD10 for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP-FcD10 activity, calcium, PPi, and pyridoxal, as well as skeletal and dental manifestations using radiography, microCT, and histomorphometry. Akp2-/- mice receiving high-dose sALP-FcD10 grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PPi, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease. Enzyme replacement using a bone-targeted, recombinant form of human TNALP prevents infantile HPP in Akp2-/- mice.

  1. Replacement of the heavily calcified ascending aorta in aortic valve replacement.

    PubMed

    Matsumoto, Kazuhisa; Hisashi, Yosuke; Imoto, Yutaka

    2015-03-01

    A totally calcified ascending aorta prevents aortic crossclamping and aortotomy during aortic valve replacement, and replacement of the ascending aorta is a valid option in these cases. We describe a simple technique for calcified ascending aorta replacement using the Cavitron Ultrasonic Surgical Aspirator. This can be used in aortic endarterectomy for removal of the calcified plaque in the anastomotic part.

  2. Aortic valve replacement with the Nicks annulus enlargement procedure 12 years after mitral valve replacement.

    PubMed

    Kazama, S; Kurata, A; Yamashita, Y

    1999-10-01

    An aortic valve replacement was successfully performed employing the Nicks annulus enlargement procedure in a case of aortic valve stenosis with small annulus 12 years after mitral valve replacement. Previous mitral valve replacement does not preclude feasibility of the Nicks procedure.

  3. Cryotherapy following total knee replacement.

    PubMed

    Adie, Sam; Kwan, Amy; Naylor, Justine M; Harris, Ian A; Mittal, Rajat

    2012-09-12

    Total knee replacement (TKR) is a common intervention for patients with end-stage osteoarthritis of the knee. Post-surgical management may include cryotherapy. However, the effectiveness of cryotherapy is unclear. To evaluate the acute (within 48 hours) application of cryotherapy following TKR on pain, blood loss and function. We searched the Cochrane Database of Systematic Reviews, CENTRAL, DARE, HTA Database, MEDLINE, EMBASE, CINAHL, PEDro and Web of Science on 15th March 2012. Randomised controlled trials or controlled clinical trials in which the experimental group received any form of cryotherapy, and was compared to any control group following TKR indicated for osteoarthritis. Two reviewers independently selected trials for inclusion. Disagreements were discussed and resolved involving a third reviewer if required. Data were then extracted and the risk of bias of trials assessed. Main outcomes were blood loss, visual analogue score (VAS) pain, adverse events, knee range of motion, transfusion rate and knee function. Secondary outcomes were analgesia use, knee swelling, length of hospital stay, quality of life and activity level. Effects of interventions were estimated as mean differences (MD), standardised mean differences (SMD) or given as risk ratios (RR), with 95% confidence intervals (CI). Meta-analyses were performed using the inverse variance method and pooled using random effects. Eleven randomised trials and one controlled clinical trial involving 809 participants met the inclusion criteria. There is very low quality evidence from 10 trials (666 participants) that cryotherapy has a small benefit on blood loss (SMD -0.46, 95% CI, -0.84 to -0.08), equivalent to 225mL less blood loss in cryotherapy group (95% CI, 39 to 410mL). This benefit may not be clinically significant. There was very low quality evidence from four trials (322 participants) that cryotherapy improved visual analogue score pain at 48 hours (MD = -1.32 points on a 10 point scale, 95% CI

  4. Adenovirus Replaces Mitotic Checkpoint Controls

    PubMed Central

    Turner, Roberta L.; Groitl, Peter; Dobner, Thomas

    2015-01-01

    ABSTRACT Infection with adenovirus triggers the cellular DNA damage response, elements of which include cell death and cell cycle arrest. Early adenoviral proteins, including the E1B-55K and E4orf3 proteins, inhibit signaling in response to DNA damage. A fraction of cells infected with an adenovirus mutant unable to express the E1B-55K and E4orf3 genes appeared to arrest in a mitotic-like state. Cells infected early in G1 of the cell cycle were predisposed to arrest in this state at late times of infection. This arrested state, which displays hallmarks of mitotic catastrophe, was prevented by expression of either the E1B-55K or the E4orf3 genes. However, E1B-55K mutant virus-infected cells became trapped in a mitotic-like state in the presence of the microtubule poison colcemid, suggesting that the two viral proteins restrict entry into mitosis or facilitate exit from mitosis in order to prevent infected cells from arresting in mitosis. The E1B-55K protein appeared to prevent inappropriate entry into mitosis through its interaction with the cellular tumor suppressor protein p53. The E4orf3 protein facilitated exit from mitosis by possibly mislocalizing and functionally inactivating cyclin B1. When expressed in noninfected cells, E4orf3 overcame the mitotic arrest caused by the degradation-resistant R42A cyclin B1 variant. IMPORTANCE Cells that are infected with adenovirus type 5 early in G1 of the cell cycle are predisposed to arrest in a mitotic-like state in a p53-dependent manner. The adenoviral E1B-55K protein prevents entry into mitosis. This newly described activity for the E1B-55K protein appears to depend on the interaction between the E1B-55K protein and the tumor suppressor p53. The adenoviral E4orf3 protein facilitates exit from mitosis, possibly by altering the intracellular distribution of cyclin B1. By preventing entry into mitosis and by promoting exit from mitosis, these adenoviral proteins act to prevent the infected cell from arresting in a

  5. What Is a Gene? (For Kids)

    MedlinePlus

    ... tested is replacing sick genes with healthy ones. Gene therapy trials — where the research is tested on people — and ... ON THIS TOPIC How to Deal With Hemophilia What's the Right Weight for Me? Do You ...

  6. Anterior cruciate ligament replacement: a review.

    PubMed

    Silver, F H; Tria, A J; Zawadsky, J P; Dunn, M G

    1991-01-01

    The anterior cruciate ligament (ACL) is the major intra-articular mechanical element that limits motion of the tibia with respect to the femur. It is a multi-fasciculated structure composed of crimped aligned collagen fibers. The purpose of this paper is to review the literature on ACL structure and mechanical properties in an effort to stimulate the development of a new generation of more effective replacement devices. Replacement of the ACL is achieved using biologic and synthetic grafts. Biologic grafts include illiotibial band, semitendinosus and gracilis tendons, patellar tendon, and meniscus. Bone-patellar-bone complexes used to replace the ACL are revascularized and ultimately replaced by neo-ligament. Synthetic implants including the Integraft, Leads-Keio ligament, Gore-Tex¿ ligament and Kennedy Ligament Augmentation Device (LAD) have either not been approved or approved by the FDA for limited use as a replacement for the ACL. The Kennedy LAD has been found to increase the strength of autogenous tissue during revascularization. Based on the success of autografts and the Kennedy LAD, we conclude that the next generation of ACL replacement devices will consist of a scaffold and a biodegradable augmentation device. The scaffold will have a structure that mimics the normal ACL as well as stimulates revascularization and healing. A biodegradable augmentation device will be employed to mechanically reinforce the scaffold without stress shielding the neo-ligament. By combining the advantages of autografts and a biodegradable augmentation device, a new generation of ACL replacements will be achieved.

  7. Fibrinogen replacement therapy for congenital fibrinogen deficiency.

    PubMed

    Bornikova, L; Peyvandi, F; Allen, G; Bernstein, J; Manco-Johnson, M J

    2011-09-01

    This review of published studies was conducted to derive data on patients with congenital fibrinogen deficiency (CFD), including dosing of fibrinogen replacement therapy, outcome, and adverse events, either temporally related or distant to fibrinogen replacement, in order to assist clinicians in developing treatment plans for patients with CFD. A systematic review was performed of case reports identified by a MEDLINE search between 1961 and 2010. Eligible studies included subjects with a diagnosis of CFD who received fibrinogen replacement. An attempt was made to extract dose, frequency, duration, hemostatic efficacy and adverse events such as thrombosis or allergic reactions. Reported thrombotic events distant from fibrinogen replacement were also recorded. From 104 papers reviewed, a total of 50 cases were identified: afibrinogenemia (35), hypofibrinogenemia (6), and dysfibrinogenemia (9). Fibrinogen replacement therapy was generally effective in preventing or treating bleeding in doses adequate to achieve and maintain fibrinogen activity above 50-100 mg dL(-1) (non-surgical and obstetric use) or 100-200 mg dL(-1) (surgical prophylaxis). Increased fibrinogen clearance was observed with massive hemorrhage, major surgery, and advanced pregnancy. Obstetric outcomes were optimized when fibrinogen replacement was initiated prior to conception. Uncontrolled hemorrhage, allergic reactions and antibody formation were rare events. However, thromboses, both related and unrelated to fibrinogen replacement, occurred in 15 of 50 (30%) patients overall, and in eight of 12 (67%) adult non-obstetric patients with afibrinogenemia. Published fibrinogen replacement regimens are presented for 50 CFD patients. Fibrinogen replacement therapy requires careful monitoring of fibrinogen levels. Afibrinogenemia is associated with thromboembolic complications with or without treatment. © 2011 International Society on Thrombosis and Haemostasis.

  8. Conceptual Design Plan SM-43 Replacement Project

    SciTech Connect

    University of California, Los Alamos National Laboratory, SCC Project Office

    2000-11-01

    The Los Alamos National Laboratory Conceptual Design Plan for the SM-43 Replacement Project outlines plans for replacing the SM-43 Administration Building. Topics include the reasons that replacement is considered a necessity; the roles of the various project sponsors; and descriptions of the proposed site and facilities. Also covered in this proposal is preliminary information on the project schedule, cost estimates, acquisition strategy, risk assessment, NEPA strategy, safety strategy, and safeguards and security. Spreadsheets provide further detail on space requirements, project schedules, and cost estimates.

  9. Replacement solvents for use in chemical synthesis

    DOEpatents

    Molnar, Linda K.; Hatton, T. Alan; Buchwald, Stephen L.

    2001-05-15

    Replacement solvents for use in chemical synthesis include polymer-immobilized solvents having a flexible polymer backbone and a plurality of pendant groups attached onto the polymer backbone, the pendant groups comprising a flexible linking unit bound to the polymer backbone and to a terminal solvating moiety. The polymer-immobilized solvent may be dissolved in a benign medium. Replacement solvents for chemical reactions for which tetrahydrofuran or diethyl may be a solvent include substituted tetrahydrofurfuryl ethers and substituted tetrahydro-3-furan ethers. The replacement solvents may be readily recovered from the reaction train using conventional methods.

  10. Beating-heart Mitral Valve Chordal Replacement

    PubMed Central

    Laing, Genevieve; Dupont, Pierre E.

    2011-01-01

    Replacing open-heart surgical procedures with beating-heart interventions substantially decreases the trauma and risk of a procedure. One of the most challenging procedures to perform on the beating heart is valve repair. To address this need, this paper proposes a tool for replacing mitral valve chordae to correct regurgitation. The chordae is secured to the papillary muscle and leaflet using NiTi tissue anchors that also incorporate an internal adjustment mechanism to enable initial adjustment as well as subsequent readjustment of chordae length. Efficacy of the proposed tool for chordae replacement and reduction of regurgitation was demonstrated in an ex-vivo heart simulator. PMID:22254843

  11. Gyrator-type circuits replace ungrounded inductors

    NASA Technical Reports Server (NTRS)

    Deboo, G. J.

    1968-01-01

    Gyrator circuits using only transistors, capacitors, and resistors which can replace both grounded and ungrounded inductors have been developed to permit complete microminiaturization of circuitry by integration of the components.

  12. Total ankle replacement. Design evolution and results.

    PubMed

    van den Heuvel, Alexander; Van Bouwel, Saskia; Dereymaeker, Greta

    2010-04-01

    The ankle joint has unique anatomical, biomechanical and cartilaginous structural characteristics that allow the joint to withstand the very high mechanical stresses and strains over years. Any minor changes to any of these features predispose the joint to osteoarthritis. Total ankle replacement (TAR) is evolving as an alternative to ankle arthrodesis for the treatment of end-stage ankle osteoarthritis. Initial implant designs from the early 1970s had unacceptably high failure and complication rates. As a result many orthopaedic surgeons have restricted the use of TAR in favour of ankle arthrodesis. Long term follow-up studies following ankle arthrodesis show risks of developing adjacent joint osteoarthritis. Therefore research towards a successful ankle replacement continues. Newer designs and longer-term outcome studies have renewed the interest in ankle joint replacement. We present an overview of the evolution, results and current concepts of total ankle replacement.

  13. Replacement Saltwell Pumping System Document Bibliography

    SciTech Connect

    BELLOMY, J.R.

    2000-12-07

    This document bibliography is prepared to identify engineering documentation developed during the design of the Replacement Saltwell Pumping System. The bibliography includes all engineering supporting documents and correspondence prepared prior to the deployment of the system in the field. All documents referenced are available electronically through the Records Management Information System (RMIS). Major components of the Replacement Saltwell Pumping System include the Sundyne Canned Motor Pump, the Water Filter Skid, the Injection Water Skid and the Backflow Preventer Assembly. Drawing H-14-104498 provides an index of drawings (fabrication details, P&IDs, etc.) prepared to support development of the Replacement Saltwell Pumping System. Specific information pertaining to new equipment can be found in Certified Vendor Information (CVI) File 50124. This CVI file has been established specifically for new equipment associated with the Replacement Saltwell Pumping System.

  14. Minimally Invasive Transcatheter Aortic Valve Replacement (TAVR)

    MedlinePlus Videos and Cool Tools

    Watch a Broward Health surgeon perform a minimally invasive Transcatheter Aortic Valve Replacement (TAVR) Click Here to view the BroadcastMed, Inc. Privacy Policy and Legal Notice © 2017 BroadcastMed, Inc. ...

  15. 47 CFR 13.17 - Replacement license.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL COMMERCIAL RADIO OPERATORS General § 13.17 Replacement... Operator License, Marine Radio Operator Permit, First Class Radiotelegraph Operator's Certificate, Second... Radio Operator's License, Restricted GMDSS Radio Operator License, GMDSS Radio Maintainer's License,...

  16. Assistance with Obtaining a Replacement Card Form

    EPA Pesticide Factsheets

    Form to be submitted to Section 608 Technician Certification Program Manager if a technician wants to replace a certification card, and the record of the original certification is in the data submitted by companies that have gone out of business.

  17. Renal replacement therapy for acute renal failure.

    PubMed

    Macedo, E; Bouchard, J; Mehta, R L

    2009-09-01

    Renal replacement therapy became a common clinical tool to treat patients with severe acute kidney injury (AKI) since the 1960s. During this time dialytic options have expanded considerably; biocompatible membranes, bicarbonate dialysate and dialysis machines with volumetric ultrafiltration control have improved the treatment for acute kidney injury. Along with advances in methods of intermittent hemodialysis, continuous renal replacement therapies have gained widespread acceptance in the treatment of dialysis-requiring AKI. However, many of the fundamental aspects of the renal replacement treatment such as indication, timing of dialytic intervention, and choice of dialysis modality are still controversial and may influence AKI patient's outcomes. This review outlines current concepts in the use of dialysis techniques for AKI and suggests an approach for selecting the optimal method of renal replacement therapy.

  18. Total ankle replacement - surgical treatment and rehabilitation.

    PubMed

    Prusinowska, Agnieszka; Krogulec, Zbigniew; Turski, Piotr; Przepiórski, Emil; Małdyk, Paweł; Księżopolska-Orłowska, Krystyna

    2015-01-01

    Functions of the ankle joint are closely connected with the gait and ability to maintain an upright position. Degenerative lesions of the joint directly contribute to postural disorders and greatly restrict propulsion of the foot, thus leading to abnormal gait. Development of total ankle replacement is connected with the use of the method as an efficient treatment of joint injuries and continuation of achievements in hip and knee surgery. The total ankle replacement technique was introduced as an alternative to arthrodesis, i.e. surgical fixation, which made it possible to preserve joint mobility and to improve gait. Total ankle replacement is indicated in post-traumatic degenerative joint disease and joint destruction secondary to rheumatoid arthritis. In this paper, total ankle replacement and various types of currently used endoprostheses are discussed. The authors also describe principles of early postoperative rehabilitation as well as rehabilitation in the outpatient setting.

  19. Aortic valve replacement in rheumatoid aortic incompetence.

    PubMed Central

    Devlin, A B; Goldstraw, P; Caves, P K

    1978-01-01

    Rheumatoid aortic valve disease is uncommon. and there are few reports of valve replacement in this condition. Aortic valve replacement and partial pericardiectomy was performed in a patient with acute rheumatoid aortitis and aortic incompetence. Previous reports suggest that any patient with rheumatoid arthritis who develops cardiac symptoms should be carefully assessed for surgically treatable involvement of the pericardium or heart valves. Images PMID:725829

  20. Testosterone replacement in older men and women.

    PubMed

    Morley, J E

    2001-01-01

    This article examines in detail the present state of the art concerning androgen deficiency in aging males. There is increasing evidence that testosterone replacement in hypogonadal older males can result in an improvement in quality of life. The major effects of testosterone are on libido, muscles, bone, and cognition. Less information is available concerning the role of testosterone in postmenopausal women, but testosterone replacement may have a role to play in treating disorders of libido and the sarcopenia that occurs at menopause.

  1. Corticosteroid replacement in critically ill patients.

    PubMed

    Jacobi, Judith

    2006-04-01

    This review addresses the use of corticosteroid replacement in critically ill patients. Low-dose corticosteroid replacement for critically ill patients with septic shock has been shown to reduce the duration of vasopressor-dependent shock, to shorten ICU length of stay, and, in recent trials, to reduce mortality. Numerous questions remain to be fully answered about patient selection, corticotropin-stimulation testing methods, and interpretation of results.

  2. Nuclear Weapons: The Reliable Replacement Warhead Program

    DTIC Science & Technology

    2005-06-23

    The Reliable Replacement Warhead Program Updated June 23, 2005 Jonathan Medalia Specialist in National Defense Foreign Affairs, Defense, and Trade...23 JUN 2005 2. REPORT TYPE 3. DATES COVERED 00-00- 2005 to 00-00- 2005 4. TITLE AND SUBTITLE Nuclear Weapons: The Reliable Replacement Warhead...Subcommittee, Strategic Forces and Nuclear Weapons Issues in Review of the Defense Authorization Request for Fiscal Year 2006, Apr. 4, 2005 , pp. 15-16

  3. Annual report for Ion Replacement Program

    SciTech Connect

    Tomczuk, Z.; Willit, J.L.; Fischer, A.K.

    1993-07-01

    Ion replacement electrorefining is an innovative electrochemical approach to purifying and separating metals. This approach overcomes the shortcomings of conventional electrorefining and has the potential for processing a wider range of metals and metal halide salts. Salt waste is also minimized with this approach. The key element of ion replacement electrorefining is the ion replacement electrode. This electrode allows a decoupling of the electrotransport process into two separate steps, anodic dissolution and cathodic deposition. Three key accomplishments described in this report that demonstrate the feasibility of ion replacement electrorefining are: (1) we have identified a suitable sodium/{beta}{double_prime}-alumina/molten salt electrolyte system that functions reproducibly at 723 K, (2) we have oxidized and deposited dysprosium, lanthanum, uranium, and titanium by using a sodium ion replacement electrode. In several experiments, an actual separation of dysprosium and lanthanum was observed, and (3) we have identified a metal alloy, Li{sub x}Sb, as an alternative ion replacement electrode. The next stage in the program is the design, construction, and testing of a laboratory-scale electrorefiner. Follow-on development with funding from industrial and federal sponsors is being pursued.

  4. A replaceable reflective film for solar concentrators

    SciTech Connect

    Not Available

    1991-09-01

    The 3M Company manufactures a silvered acrylic film called ECP-305 that is regarded as the preferred reflective film for use on stretched-membrane heliostats. However, ECP-305 will degrade in time, due to both corrosion of the silver layer and delamination at the film's silver-to-acrylic interface, and will eventually need to be replaced. 3M uses a very aggressive adhesive on this film, and once it is laminated, replacement is very difficult. The purpose of this investigation was the development of a replaceable reflector, a reflective film that can be easily removed and replaced. A replaceable reflector was successfully configured by laminating ECP-305 to the top surface of a smooth, dimensionally stable polymer film, with a removable adhesive applied to the underside of the polymer film. Several stages of screening and testing led to the selection of a 0.010-inch thick polycarbonate (GE 8030) as the best polymer film and a medium tack tape (3M Y-9425) was selected as the best removable adhesive. To demonstrate the feasibility of the replaceable reflector concept and to provide a real-time field test, the chosen construction was successfully applied to the 50-m{sup 2} SKI heliostat at the Central Receiver Test Facility at Sandia National Laboratories in Albuquerque. 4 refs., 13 figs., 7 tabs.

  5. Galvanic Replacement of the Liquid Metal Galinstan.

    PubMed

    Hoshyargar, Faegheh; Crawford, Jessica; O'Mullane, Anthony P

    2017-02-01

    The galvanic replacement reaction is a highly versatile approach for the creation of a variety of nanostructured materials. However, the majority of reports are limited to the replacement of metallic nanoparticles or metal surfaces. Here we extend this elegant approach and describe the galvanic replacement of the liquid metal alloy galinstan with Ag and Au. This is achieved at a macrosized droplet to create a liquid metal marble that comprises a liquid metal core and a solid metal shell, whereby the morphology of the outer shell is determined by the concentration of metallic ions used in the solution during the galvanic replacement process. In principle, this allows one to recover precious metal ions from solution in their metallic form, which are immobilized on the liquid metal and therefore easy to recover. The reaction is also undertaken at liquid metal microdroplets created via sonication to produce Ag- and Au-based galinstan nanorice particles. These materials are characterized with SEM, XRD, TEM, SAED, EDX, XPS, UV-visible spectroscopy, and open-circuit potential versus time experiments to understand the galvanic replacement process. Finally, the nanosized materials are investigated for their catalytic activity toward the reduction of methylene blue in the presence of sodium borohydride. This approach illustrates a new avenue of research for the galvanic replacement process and, in principle, could be applied to many more systems.

  6. [Replacement of tracheo-esophageal Provox prosthesis].

    PubMed

    Biacabe, B; Laccourreye, O; Ménard, M; Hans, S; Brasnu, D

    2000-02-01

    To compare anesthesic techniques used between 1992 and 1997 at Laënnec Hospital for replacement by tracheo-esophageal Provox prosthesis: local and general anesthesia. Theoretical financial cost for replacement was estimated according to anaesthetic techniques. Provox in situ lifetime was calculated in 58 patients who underwent 115 and 49 replacements under general and local anaesthesia respectively. Age, sex, surgical and radiotherapy backgrounds, complications and anaesthetic techniques were studied as potential factors correlated with Provox in situ lifetime. Theoretical financial cost for replacement was estimated according to anaesthetic techniques. In 1992, 12% of Provox prosthesis were inserted under local anaesthesia and 54% in 1997. Provox in situ lifetime was either not influenced by anaesthetic techniques or other factors under analysis. The theoretical financial cost was estimated at 14, 341 FFrs and 6,048 FFrs for replacement under general and local anaesthesia respectively. Due to increased control of health care costs, we advocated local anaesthesia for Provox prosthesis replacement if control endoscopy is not required.

  7. Rapid, efficient and precise allele replacement in the fission yeast Schizosaccharomyces pombe

    PubMed Central

    Gao, Jun; Kan, Fengling; Wagnon, Jacy L.; Storey, Aaron J.; Protacio, Reine M.; Davidson, Mari K.; Wahls, Wayne P.

    2013-01-01

    Gene targeting provides a powerful tool to modify endogenous loci to contain specific mutations, insertions and deletions. Precise allele replacement, with no other chromosomal changes (e.g., insertion of selectable markers or heterologous promoters), maintains physiologically relevant context. Established methods for precise allele replacement in fission yeast employ two successive rounds of transformation and homologous recombination and require genotyping at each step. The relative efficiency of homologous recombination is low and a high rate of false positives during the second round of gene targeting further complicates matters. We report that pop-in, pop-out allele replacement circumvents these problems. We present data for 39 different allele replacements, involving simple and complex modifications at seven different target loci, that illustrate the power and utility of the approach. We also developed and validated a rapid, efficient process for precise allele replacement that requires only one round each of transformation and genotyping. We show that this process can be applied in population scale to an individual target locus, without genotyping, to identify clones with an altered phenotype (targeted forward genetics). It is therefore suitable for saturating, in situ, locus-specific mutation screens (e.g., of essential or non-essential genes and regulatory DNA elements) within normal chromosomal context. PMID:24026504

  8. The Scandinavian Total Ankle Replacement and the ideal biomechanical requirements of ankle replacements.

    PubMed

    Robati, Shibby; Salih, Alan; Ghosh, Koushik; Vinayakam, Parthiban

    2016-03-01

    The complex anatomy of the articular bone surfaces, ligaments, tendon attachments and muscles makes the ankle joint difficult to replicate in prosthetic replacements. Ever since the early 1970s, which saw the dawn of the first total ankle replacements, there have been numerous other attempts at replicating the joint, often with poor clinical outcomes. The anatomy of the ankle is discussed, followed by evidence of the normal ankle biomechanics and the ideal requirements of an ankle replacement. We focus on the Scandinavian Total Ankle Replacement and evaluate whether these requirements have been met.

  9. The Scandinavian Total Ankle Replacement and the ideal biomechanical requirements of ankle replacements

    PubMed Central

    Robati, Shibby; Salih, Alan; Ghosh, Koushik; Vinayakam, Parthiban

    2016-01-01

    The complex anatomy of the articular bone surfaces, ligaments, tendon attachments and muscles makes the ankle joint difficult to replicate in prosthetic replacements. Ever since the early 1970s, which saw the dawn of the first total ankle replacements, there have been numerous other attempts at replicating the joint, often with poor clinical outcomes. The anatomy of the ankle is discussed, followed by evidence of the normal ankle biomechanics and the ideal requirements of an ankle replacement. We focus on the Scandinavian Total Ankle Replacement and evaluate whether these requirements have been met. PMID:26955224

  10. Simple and rapid direct cloning and heterologous expression of natural product biosynthetic gene cluster in Bacillus subtilis via Red/ET recombineering

    PubMed Central

    Liu, Qingshu; Shen, Qiyao; Bian, Xiaoying; Chen, Hanna; Fu, Jun; Wang, Hailong; Lei, Ping; Guo, Zhaohui; Chen, Wu; Li, Dingjun; Zhang, Youming

    2016-01-01

    Heterologous expression of biosynthetic pathways is an important way to research and discover microbial natural products. Bacillus subtilis is a suitable host for the heterologous production of natural products from bacilli and related Firmicutes. Existing technologies for heterologous expression of large biosynthetic gene clusters in B. subtilis are complicated. Herein, we present a simple and rapid strategy for direct cloning based heterologous expression of biosynthetic pathways in B. subtilis via Red/ET recombineering, using a 5.2 kb specific direct cloning vector carrying homologous sequences to the amyE gene in B. subtilis and CcdB counterselection marker. Using a two-step procedure, two large biosynthetic pathways for edeine (48.3 kb) and bacillomycin (37.2 kb) from Brevibacillus brevis X23 and B. amyloliquefaciens FZB42, respectively, were directly cloned and subsequently integrated into the chromosome of B. subtilis within one week. The gene cluster for bacillomycin was successfully expressed in the heterologous host, although edeine production was not detectable. Compared with similar technologies, this method offers a simpler and more feasible system for the discovery of natural products from bacilli and related genera. PMID:27687863

  11. 42 CFR 433.117 - Initial approval of replacement systems.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... and Information Retrieval Systems § 433.117 Initial approval of replacement systems. (a) A replacement... retrieval system. (b) The agency must submit a APD that includes— (1) The date the replacement system...

  12. Hip or knee replacement - before - what to ask your doctor

    MedlinePlus

    ... PA: Elsevier Mosby; 2012:chap 7. Read More Hip joint replacement Hip pain Knee joint replacement Knee pain ... joint replacement - discharge Taking care of your new hip joint Review Date 3/5/2015 Updated by: C. ...

  13. Hip or knee replacement - after - what to ask your doctor

    MedlinePlus

    ... PA: Elsevier Mosby; 2012:chap 7. Read More Hip joint replacement Hip pain Knee joint replacement Knee pain ... joint replacement - discharge Taking care of your new hip joint Review Date 3/5/2015 Updated by: C. ...

  14. Kittiwakes strategically reduce investment in replacement clutches

    USGS Publications Warehouse

    Gasparini, J.; Roulin, A.; Gill, V.A.; Hatch, Shyla A.; Boulinier, T.

    2006-01-01

    Many life-history traits are expressed interactively in life, but to a varying extent on different occasions. Changes in trait expression can be accounted for by differences in the quality of the environment ('environmental constraint' hypothesis) or by strategic adjustments, if the relative contribution of the trait to fitness varies with time ('strategic allocation' hypothesis). In birds, egg production is lower in replacement clutches than in first clutches, but it is unknown whether this reduction results from an environmental constraint (e.g. food being less available at the time when the replacement clutch is produced) or from a strategic allocation of resources between the two breeding attempts. To distinguish between these two hypotheses, we performed an experiment with black-legged kittiwakes (Rissa tridactyla). Pairs were either food-supplemented or not before the first clutch was laid onwards and we induced them to produce a replacement clutch by removing eggs once when the first clutch was complete. As predicted by the 'strategic allocation' hypothesis, egg production of food-supplemented and non-food-supplemented birds decreased between first and replacement clutches. This suggests that kittiwakes strategically reduce investment in egg production for their replacement clutches compared to first clutches. ?? 2006 The Royal Society.

  15. Tracheal replacement with an aortic autograft.

    PubMed

    Azorin, Jacques F; Bertin, Francois; Martinod, Emmanuel; Laskar, Marc

    2006-02-01

    Tracheal replacement after extensive resection remains a challenge for the thoracic surgeon. We propose an innovative solution: the use of an aortic autograft. After an experimental work on animals with aortic autografts and allografts [Martinod E, Seguin A, Pfeuty K, Fornes P, Kambouchner M, Azorin JF, Carpentier AF. Long-term evaluation of the replacement of the trachea with an autologous aortic graft. Ann Thorac Surg 2003;75(5):1572-8; Martinod E, Seguin A, Holder-Espinasse M, Kambouchner M, Duterque-Coquillaud M, Azorin JF, Carpentier AF. Tracheal regeneration following tracheal replacement with an allogenic aorta. Ann Thorac Surg 2005;79(3):942-8], we present the first human case of long tracheal replacement with an aortic autograft. In this case we replaced 7 cm of a tumoral trachea using an aortic infra-renal autograft supported by a silicone stent. The early postoperative course was uneventful. The stent was removed at three months. The patient died at six months from an acute pulmonary infection without any sign of anastomosis leakage or graft rupture. A new field of clinical study has to be investigated.

  16. Rational optimization of tolC as a powerful dual selectable marker for genome engineering

    PubMed Central

    Gregg, Christopher J.; Lajoie, Marc J.; Napolitano, Michael G.; Mosberg, Joshua A.; Goodman, Daniel B.; Aach, John; Isaacs, Farren J.; Church, George M.

    2014-01-01

    Selection has been invaluable for genetic manipulation, although counter-selection has historically exhibited limited robustness and convenience. TolC, an outer membrane pore involved in transmembrane transport in E. coli, has been implemented as a selectable/counter-selectable marker, but counter-selection escape frequency using colicin E1 precludes using tolC for inefficient genetic manipulations and/or with large libraries. Here, we leveraged unbiased deep sequencing of 96 independent lineages exhibiting counter-selection escape to identify loss-of-function mutations, which offered mechanistic insight and guided strain engineering to reduce counter-selection escape frequency by ∼40-fold. We fundamentally improved the tolC counter-selection by supplementing a second agent, vancomycin, which reduces counter-selection escape by 425-fold, compared colicin E1 alone. Combining these improvements in a mismatch repair proficient strain reduced counter-selection escape frequency by 1.3E6-fold in total, making tolC counter-selection as effective as most selectable markers, and adding a valuable tool to the genome editing toolbox. These improvements permitted us to perform stable and continuous rounds of selection/counter-selection using tolC, enabling replacement of 10 alleles without requiring genotypic screening for the first time. Finally, we combined these advances to create an optimized E. coli strain for genome engineering that is ∼10-fold more efficient at achieving allelic diversity than previous best practices. PMID:24452804

  17. Random transposon vectors pUTTns for the markerless integration of exogenous genes into gram-negative eubacteria chromosomes.

    PubMed

    Li, Rong; Wang, Guangli; Shen, Bin; Wang, Rong; Song, Yao; Li, Shunpeng; Jiang, Jiandong

    2009-11-01

    A set of random transposon vectors pUTTns that facilitates the markerless integration of new functions into the chromosome of gram-negative bacteria has been developed. The vectors, which are derived from mini-Tn5 transposons, are located on a R6K-based suicide delivery plasmid that provides the IS50(R) transposase tnp gene in cis, but they are external to the mobile element. The vectors' conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor. Internal to the mini-Tn5 element is a cassette that contains a selectable antibiotic resistance marker (kanamycin, chloramphenicol, or tetracycline resistance gene), a counter-selectable marker (sacB), a 430-bp repeat of the sacB gene 3' end acted as the directly-repeated (DR) sequence, and modified multiple cloning sites (MCS). After two total rounds of transposon integration and recombination between the two DRs, only the exogenous DNA inserted into the MCS (passenger genes) and a single 430-bp scar sacBDR fragment remained in the chromosome after excision. The utility of these vectors was demonstrated by integrating the organophosphorus insecticide hydrolase gene (mpd) into the chromosome of Escherichia, Pseudomonas, Sphingomonas, and Paracoccus species. Sequential integration of another organophosphorus insecticide hydrolase gene (oph) into the previously engineered bacteria, without bringing any selectable markers, was also successful. These engineered bacteria were relatively stable. Cell viability and original degrading characteristics were not affected compared with the original recipients. This shows that the developed system is very useful for the markerless integration of exogenous genes into the chromosome of gram-negative eubacteria.

  18. Gene therapy for blindness.

    PubMed

    Sahel, José-Alain; Roska, Botond

    2013-07-08

    Sight-restoring therapy for the visually impaired and blind is a major unmet medical need. Ocular gene therapy is a rational choice for restoring vision or preventing the loss of vision because most blinding diseases originate in cellular components of the eye, a compartment that is optimally suited for the delivery of genes, and many of these diseases have a genetic origin or genetic component. In recent years we have witnessed major advances in the field of ocular gene therapy, and proof-of-concept studies are under way to evaluate the safety and efficacy of human gene therapies. Here we discuss the concepts and recent advances in gene therapy in the retina. Our review discusses traditional approaches such as gene replacement and neuroprotection and also new avenues such as optogenetic therapies. We conjecture that advances in gene therapy in the retina will pave the way for gene therapies in other parts of the brain.

  19. Replacement of charcoal sorbent in the VOST

    SciTech Connect

    Johnson, L.D.; Fuerst, R.G.; Foster, A.L.; Bursey, J.T.

    1993-01-01

    EPA Method 0030, the Volatile Organic Sampling Train (VOST), for sampling volatile organics from stationary sources, specifies the use of petroleum-base charcoal in the second sorbent tube. Charcoal has proven to be a marginal performer as a sampling sorbent, partly due to inconsistency in analyte recovery. In addition, commercial availability of petroleum charcoal for VOST tubes has been variable. Lack of data on comparability and variability of charcoals for VOST application has created uncertainty when other charcoals are substituted. Five potential sorbent replacements for charcoal in Method 0030 were evaluated along with a reference charcoal. Two of the sorbents tested, Ambersorb XE-340 and Tenax GR, did not perform well enough to qualify as replacements. Three candidates, Anasorb 747, Carbosieve S-III and Kureha Beaded Activated Charcoal, performed adequately, and produced statistically equivalent results. Anasorb 747 appears to be an acceptable replacement for petroleum charcoal, based on a combination of performance, availability, and cost.

  20. Object replacement using dynamic proxy updates

    NASA Astrophysics Data System (ADS)

    Menon, S.; LeBlanc, R. J., Jr.

    1994-09-01

    In a distributed environment, a client program bound to a server fails when the server changes (possibly due to the server being relocated, replicated or reconfigured). In this paper, we describe the design of an object-replacement scheme in a client-server environment. Our design addresses the problem of replacing a server and transparently updating the client handle so that no service interruption is experienced by the client. The programming environment, on which this work is based, provides passive shared objects, threads, and invocations as the building blocks for distributed applications. Our design takes into account the potential concurrency within clients and servers. Our task was relatively simplified by building the replacement mechanisms on top of an asynchronous event notification facility that handles events on a per-application basis, as opposed to a per-task or per-thread basis.

  1. HP replacement program increases efficiency, protection

    SciTech Connect

    1997-10-01

    For more than 50 years, compression equipment along the 2,000-mile Tennessee Gas Pipeline has been helping to supply natural gas needs for the Northeast. But increasing demand and a need for more environmentally safe equipment mean a major replacement program for the compressor stations that make the natural gas transmission possible. Today it is one of the longest gas pipelines in the world, carrying more than 1 Bcf/d of natural gas. New compression equipment is being installed to boost efficiency and meet more stringent environmental standards. In 1993, Tenneco Energy, purchased by El Paso Energy in December 1996, initiated a Horsepower Replacement Program intended to replace older, inefficient technology with more advanced equipment. A major objective was to improve operational effectiveness and to reduce harmful nitrogen oxide and carbon monoxide emissions by converting much of the machinery to electric-driven compression equipment.

  2. Double Valve Replacement for Lupus Valvulitis

    PubMed Central

    Ferraris, Victor A.; Hawksley, Vaughn C.; Rabinowitz, Max; Coyne, Carolyn M.; Sullivan, Thomas J.; Sprague, Merle S.

    1990-01-01

    Hemodynamically significant lupus valvulitis, requiring valve replacement, is rare: 21 cases have been reported so far in the literature, and only 2 of these have involved double valve replacement. We describe an additional case of double valve replacement in a patient with systemic lupus erythematosus. The histopathologic and clinical features of this case suggest that valvular involvement resulted from both acute and chronic disease processes. Medical success in the treatment of systemic lupus erythematosus, especially that achieved through prolonged or high-dose steroid therapy, may cause chronic valvular disease to become a more common surgical problem. A review of the literature supports this contention. (Texas Heart Institute Journal 1990;17:56-60) Images PMID:15227191

  3. Testosterone replacement therapy and prostate health.

    PubMed

    Polackwich, A Scott; Ostrowski, Kevin A; Hedges, Jason C

    2012-12-01

    There is an emerging evolution in the understanding of the relationship between the prostate and testosterone. It has long been generally believed that with testosterone replacement therapy (TRT), increasing serum testosterone levels led to prostatic growth and worsening of voiding dysfunction and associated complications. A new theory, the Saturation Model of Testosterone and its effect on the Prostate has gained attention. This theory suggests that the prostate's response to increasing levels of serum testosterone reaches a limit beyond which there is minimal effect. This model predicts that testosterone replacement therapy occurs above this saturation point, and replacing testosterone to eugonadal levels should not worsen prostate related benign disease. We evaluated the recent published data, with an emphasis on clinical studies done within the last 3 years, for the effects of testosterone supplementation on benign prostatic disease.

  4. Dead pixel replacement in LWIR microgrid polarimeters.

    PubMed

    Ratliff, Bradley M; Tyo, J Scott; Boger, James K; Black, Wiley T; Bowers, David L; Fetrow, Matthew P

    2007-06-11

    LWIR imaging arrays are often affected by nonresponsive pixels, or "dead pixels." These dead pixels can severely degrade the quality of imagery and often have to be replaced before subsequent image processing and display of the imagery data. For LWIR arrays that are integrated with arrays of micropolarizers, the problem of dead pixels is amplified. Conventional dead pixel replacement (DPR) strategies cannot be employed since neighboring pixels are of different polarizations. In this paper we present two DPR schemes. The first is a modified nearest-neighbor replacement method. The second is a method based on redundancy in the polarization measurements.We find that the redundancy-based DPR scheme provides an order-of-magnitude better performance for typical LWIR polarimetric data.

  5. Nashville Gas treads carefully to replace pipe

    SciTech Connect

    1997-06-01

    The private gas utility, Nashville Gas, was responsible for replacing damaged or inadequate 2- and 4-inch steel gas lines beneath Music City, USA. The line replacements required either size for size or upsizing. The first choice was directional drilling, which was quickly determined to be unpractical because of rocky soil conditions. The second option was open trenching. Undoubtedly, trenching would mean having to contend with angry residents and tourists, since gas lines ran beneath yards, mature trees, sidewalks, roadways, and railways. In addition to the negative social factors, trenching would require additional funds for substantial landscaping and pavement replacement. It at all possible, a no-dig alternative was desired. Nashville Gas found Grundomat piercing tools which create a bore, then pushes pipe back through it. These same tools can simultaneously pull in pipe. These tools were customized for the Nashville project.

  6. [Update in continuous renal replacement techniques].

    PubMed

    Romero-García, M; de la Cueva-Ariza, L; Delgado-Hito, P

    2013-01-01

    Acute renal failure affects 25% of patients hospitalized in intensive care units. Despite technological advances, the mortality of these patients is still high due to its associated complications. Continuous renal replacement techniques are one of the treatments for acute renal failure because they make it possible to treat the complications and decrease mortality. The nurse's knowledge and skills regarding these techniques will be decisive for the success of the therapy. Consequently, the nurse's experience and training are key components. The objective of this article is to update the knowledge on continuous renal replacement techniques. Keeping this in mind, a review has been made of the physical and chemical principles such as diffusion and convection, among others. A description of the different continuous renal replacement techniques, a presentation of the main vascular access, and a description of the nursing cares and complications related to techniques used have also been provided.

  7. Phaser.MRage: automated molecular replacement

    SciTech Connect

    Bunkóczi, Gábor; McCoy, Airlie J.; Oeffner, Robert D.; Read, Randy J.

    2013-11-01

    The functionality of the molecular-replacement pipeline phaser.MRage is introduced and illustrated with examples. Phaser.MRage is a molecular-replacement automation framework that implements a full model-generation workflow and provides several layers of model exploration to the user. It is designed to handle a large number of models and can distribute calculations efficiently onto parallel hardware. In addition, phaser.MRage can identify correct solutions and use this information to accelerate the search. Firstly, it can quickly score all alternative models of a component once a correct solution has been found. Secondly, it can perform extensive analysis of identified solutions to find protein assemblies and can employ assembled models for subsequent searches. Thirdly, it is able to use a priori assembly information (derived from, for example, homologues) to speculatively place and score molecules, thereby customizing the search procedure to a certain class of protein molecule (for example, antibodies) and incorporating additional biological information into molecular replacement.

  8. Rapid prototyping of ossicular replacement prostheses

    NASA Astrophysics Data System (ADS)

    Ovsianikov, A.; Chichkov, B.; Adunka, O.; Pillsbury, H.; Doraiswamy, A.; Narayan, R. J.

    2007-05-01

    Materials used in ossicular replacement prostheses must demonstrate appropriate biological compatibility, acoustic transmission, stability, and stiffness properties. Prostheses prepared using Teflon ®, titanium, Ceravital and other conventional materials have demonstrated several problems, including migration, perforation of the tympanic membrane, difficulty in shaping the prostheses, and reactivity with the surrounding tissues. We have used two-photon polymerization for rapid prototyping of Ormocer ® middle-ear bone replacement prostheses. Ormocer ® surfaces fabricated using two-photon polymerization exhibited acceptable cell viability and cell growth profiles. The Ormocer ® prosthesis was able to be inserted and removed from the site of use in the frozen human head without fracture. Our results demonstrate that two-photon polymerization is able to create ossicular replacement prostheses and other medical devices with a larger range of sizes, shapes and materials than other microfabrication techniques.

  9. Swapping symbionts in spittlebugs: evolutionary replacement of a reduced genome symbiont

    PubMed Central

    Koga, Ryuichi; Moran, Nancy A

    2014-01-01

    Bacterial symbionts that undergo long-term maternal transmission experience elevated fixation of deleterious mutations, resulting in massive loss of genes and changes in gene sequences that appear to limit efficiency of gene products. Potentially, this dwindling of symbiont functionality impacts hosts that depend on these bacteria for nutrition. One evolutionary escape route is the acquisition of a novel symbiont with a robust genome and metabolic capabilities. Such an acquisition has occurred in an ancestor of Philaenus spumarius, the meadow spittlebug (Insecta: Cercopoidea), which has replaced its ancient association with the tiny genome symbiont Zinderia insecticola (Betaproteobacteria) with an association with a symbiont related to Sodalis glossinidius (Gammaproteobacteria). Spittlebugs feed exclusively on xylem sap, a diet that is low both in essential amino acids and in sugar or other substrates for energy production. The new symbiont genome has undergone proliferation of mobile elements resulting in many gene inactivations; nonetheless, it has selectively maintained genes replacing functions of its predecessor for amino-acid biosynthesis. Whereas ancient symbiont partners typically retain perfectly complementary sets of amino-acid biosynthetic pathways, the novel symbiont introduces some redundancy as it retains some pathways also present in the partner symbionts (Sulcia muelleri). Strikingly, the newly acquired Sodalis-like symbiont retains genes underlying efficient routes of energy production, including a complete TCA cycle, potentially relaxing the severe energy limitations of the xylem-feeding hosts. Although evolutionary replacements of ancient symbionts are infrequent, they potentially enable evolutionary and ecological novelty by conferring novel metabolic capabilities to host lineages. PMID:24401857

  10. Swapping symbionts in spittlebugs: evolutionary replacement of a reduced genome symbiont.

    PubMed

    Koga, Ryuichi; Moran, Nancy A

    2014-06-01

    Bacterial symbionts that undergo long-term maternal transmission experience elevated fixation of deleterious mutations, resulting in massive loss of genes and changes in gene sequences that appear to limit efficiency of gene products. Potentially, this dwindling of symbiont functionality impacts hosts that depend on these bacteria for nutrition. One evolutionary escape route is the acquisition of a novel symbiont with a robust genome and metabolic capabilities. Such an acquisition has occurred in an ancestor of Philaenus spumarius, the meadow spittlebug (Insecta: Cercopoidea), which has replaced its ancient association with the tiny genome symbiont Zinderia insecticola (Betaproteobacteria) with an association with a symbiont related to Sodalis glossinidius (Gammaproteobacteria). Spittlebugs feed exclusively on xylem sap, a diet that is low both in essential amino acids and in sugar or other substrates for energy production. The new symbiont genome has undergone proliferation of mobile elements resulting in many gene inactivations; nonetheless, it has selectively maintained genes replacing functions of its predecessor for amino-acid biosynthesis. Whereas ancient symbiont partners typically retain perfectly complementary sets of amino-acid biosynthetic pathways, the novel symbiont introduces some redundancy as it retains some pathways also present in the partner symbionts (Sulcia muelleri). Strikingly, the newly acquired Sodalis-like symbiont retains genes underlying efficient routes of energy production, including a complete TCA cycle, potentially relaxing the severe energy limitations of the xylem-feeding hosts. Although evolutionary replacements of ancient symbionts are infrequent, they potentially enable evolutionary and ecological novelty by conferring novel metabolic capabilities to host lineages.

  11. The echocardiography of replacement heart valves

    PubMed Central

    2016-01-01

    This is a practical description of how replacement valves are assessed using echocardiography. Normal transthoracic appearances including normal variants are described. The problem of differentiating normal function, patient–prosthesis mismatch and pathological obstruction in aortic replacement valves with high gradients is discussed. Obstruction and abnormal regurgitation is described for valves in the aortic, mitral and right-sided positions and when to use echocardiography in suspected infective endocarditis. The roles of transoesophageal and stress echocardiography are described and finally when other imaging techniques may be useful. PMID:27600454

  12. Valve assembly having remotely replaceable bearings

    DOEpatents

    Johnson, Evan R.; Tanner, David E.

    1980-01-01

    A valve assembly having remotely replaceable bearings is disclosed wherein a valve disc is supported within a flow duct for rotation about a pair of axially aligned bearings, one of which is carried by a spindle received within a diametral bore in the valve disc, and the other of which is carried by a bearing support block releasably mounted on the duct circumferentially of an annular collar on the valve disc coaxial with its diametrical bore. The spindle and bearing support block are adapted for remote removal to facilitate servicing or replacement of the valve disc support bearings.

  13. Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

    PubMed Central

    Li, Nan; Qin, Ting; Zhang, Xiao Lin; Huang, Bei; Liu, Zhi Xin; Xie, Hai Xia; Zhang, Jin; McBride, Mark J.

    2015-01-01

    Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ΔcslA and ΔcslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslA ΔcslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G4 and of the chondroitin lyase-deficient ΔcslA ΔcslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes. PMID:26253667

  14. Gene deletion strategy to examine the involvement of the two chondroitin lyases in Flavobacterium columnare virulence.

    PubMed

    Li, Nan; Qin, Ting; Zhang, Xiao Lin; Huang, Bei; Liu, Zhi Xin; Xie, Hai Xia; Zhang, Jin; McBride, Mark J; Nie, Pin

    2015-11-01

    Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ΔcslA and ΔcslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslA ΔcslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G4 and of the chondroitin lyase-deficient ΔcslA ΔcslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes.

  15. Dithizone Staining of Intracellular Zinc: An Unexpected and Versatile Counterscreen for Auxotrophic Marker Genes in Saccharomyces cerevisiae

    PubMed Central

    Yuan, Daniel S.

    2011-01-01

    Auxotrophic marker genes such as URA3, LEU2, and HIS3 in Saccharomyces cerevisiae have long been used to select cells that have been successfully transformed with recombinant DNA. A longstanding challenge in working with these genes is that counterselection procedures are often lacking. This paper describes the unexpected discovery of a simple plate assay that imparts a bright red stain to cells experiencing nutritional stress from the lack of a marker gene. The procedure specifically stains a zinc-rich vesicular compartment analogous to the zinc-rich secretory vesicles found in insulin-secreting pancreatic islet cells and glutamate-secreting neurons. Staining was greatly diminished in zap1 mutants, which lack a homeostatic activator of zinc uptake, and in cot1 zrc1 double mutants, which lack the two yeast homologs of mammalian vesicle-specific zinc export proteins. Only one of 93 strains with temperature-sensitive alleles of essential genes exhibited an increase in dithizone staining at its non-permissive temperature, indicating that staining is not simply a sign of growth-arrested or dying cells. Remarkably, the procedure works with most commonly used marker genes, highlights subtle defects, uses no reporter constructs or expensive reagents, requires only a few hours of incubation, yields visually striking results without any instrumentation, and is not toxic to the cells. Many potential applications exist for dithizone staining, both as a versatile counterscreen for auxotrophic marker genes and as a powerful new tool for the genetic analysis of a biomedically important vesicular organelle. PMID:21998704

  16. REPLACING SOLVENT CLEANING WITH AQUEOUS CLEANING

    EPA Science Inventory

    The report documents actions taken by Robert Bosch Corp., Charleston, SC, in replacing the cleaning solvents 1, 1, 2- trichloro-1, 2, 2-trifluoroethane (CFC-113) and trichloroethylene (TCE) with aqueous solutions. Bosch has succeeded in eliminating all their CFC-113 use and so f...

  17. REPLACING SOLVENT CLEANING WITH AQUEOUS CLEANING

    EPA Science Inventory

    The report documents actions taken by Robert Bosch Corp., Charleston, SC, in replacing the cleaning solvents 1, 1, 2- trichloro-1, 2, 2-trifluoroethane (CFC-113) and trichloroethylene (TCE) with aqueous solutions. Bosch has succeeded in eliminating all their CFC-113 use and so f...

  18. SYNERGISTIC WOOD PRESERVATIVES FOR REPLACEMENT OF CCA

    EPA Science Inventory

    The objective of this project was to evaluate the potential synergistic combinations of environmentally-safe biocides as wood preservatives. These wood preservatives could be potential replacements for the heavy-metal based CCA.

    Didecyldimethylammonium chloride [DDAC] was...

  19. 24 CFR 891.745 - Replacement reserve.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 4 2011-04-01 2011-04-01 false Replacement reserve. 891.745 Section 891.745 Housing and Urban Development REGULATIONS RELATING TO HOUSING AND URBAN DEVELOPMENT (CONTINUED) OFFICE OF THE ASSISTANT SECRETARY FOR HOUSING-FEDERAL HOUSING COMMISSIONER, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT (SECTION 8 HOUSING...

  20. 24 CFR 891.745 - Replacement reserve.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Replacement reserve. 891.745 Section 891.745 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF THE ASSISTANT SECRETARY FOR HOUSING-FEDERAL HOUSING COMMISSIONER, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT (SECTION 8 HOUSING...

  1. 24 CFR 891.745 - Replacement reserve.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 4 2012-04-01 2012-04-01 false Replacement reserve. 891.745 Section 891.745 Housing and Urban Development REGULATIONS RELATING TO HOUSING AND URBAN DEVELOPMENT (CONTINUED) OFFICE OF THE ASSISTANT SECRETARY FOR HOUSING-FEDERAL HOUSING COMMISSIONER, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT (SECTION 8 HOUSING...

  2. 25 CFR 700.53 - Dwelling, replacement.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... § 700.55. (b) May include existing dwellings for resale, new construction, modular homes, mobile homes..., his new monthly housing cost (defined at § 700.81) for the replacement dwelling does not exceed twenty... projects, tribal-sponsored housing projects and/or Commission-sponsored housing projects. ...

  3. Winged scapula after aortic valve replacement.

    PubMed

    Schmitz, Christoph; Sodian, Ralf; Witt, Thomas N; Juchem, Gerd; Lang, Nora; Bruegger, Christian; Kowalski, Christian; Reichart, Bruno

    2009-04-01

    Iatrogenic nerve lesions affecting the long thoracic nerve are very rare after a median sternotomy. Here we report on a patient who developed clinical signs of a so-called "winged scapula" after an uneventful aortic valve replacement for infective endocarditis.

  4. Probability Issues in without Replacement Sampling

    ERIC Educational Resources Information Center

    Joarder, A. H.; Al-Sabah, W. S.

    2007-01-01

    Sampling without replacement is an important aspect in teaching conditional probabilities in elementary statistics courses. Different methods proposed in different texts for calculating probabilities of events in this context are reviewed and their relative merits and limitations in applications are pinpointed. An alternative representation of…

  5. Nicotine Replacement: Effects on Postcessation Weight Gain.

    ERIC Educational Resources Information Center

    Gross, Janet; And Others

    1989-01-01

    Examined nicotine replacement effects on postcessation weight gain in smoking cessation volunteers. Randomly assigned abstinent subjects to active nicotine or placebo gum conditions for 10 weeks. Analyses revealed strong evidence for gum effect on weight gain, with active gum users gaining mean total of 3.8 pounds compared with 7.8 pounds for…

  6. Options for Replacing and Reformatting Deteriorated Materials.

    ERIC Educational Resources Information Center

    Banks, Jennifer

    This resource guide focuses on handling library materials that are too deteriorated to benefit from other preservation treatments. Hopeless case items, that would require an unreasonable amount of conservation work to fix, can be resolved through a process of reselecting items by replacing them or reformatting them. The reselection integrates…

  7. Probability Issues in without Replacement Sampling

    ERIC Educational Resources Information Center

    Joarder, A. H.; Al-Sabah, W. S.

    2007-01-01

    Sampling without replacement is an important aspect in teaching conditional probabilities in elementary statistics courses. Different methods proposed in different texts for calculating probabilities of events in this context are reviewed and their relative merits and limitations in applications are pinpointed. An alternative representation of…

  8. Recent Advances in Hydrocortisone Replacement Treatment.

    PubMed

    Mallappa, Ashwini; Debono, Miguel

    2016-01-01

    Since the first use of cortisone in patients around 65 years ago, the use of synthetic glucocorticoids has made a crucial impact on the treatment of several diseases in medicine. Although significant reductions in morbidity and mortality have occurred in patients suffering from cortisol deficiency, conventional hydrocortisone replacement treatment is still inadequate. A major limitation is that it fails to replace cortisol in a physiological manner. Cortisol has a distinct circadian rhythm and acts as a secondary messenger synchronizing the central to peripheral clocks, hence playing a key role in biological processes and the circadian timing system. Circadian misalignment has been associated with ill-health and so nonphysiological glucocorticoid treatment could explain the increased mortality rate, poor quality of life and metabolic complications in patients suffering from adrenal insufficiency. Attempts at replacing cortisol in a physiological manner have shown significant progress in the past decade with the development of modified-release formulations of hydrocortisone (Chronocort® and Plenadren®) and continuous subcutaneous hydrocortisone infusions. Initial studies investigating the use of these replacement regimens are promising, demonstrating both clinical and biochemical improvement. Larger studies are needed to determine whether this novel approach enhances long-term outcomes in both children and adults with cortisol deficiency. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply. Published by S. Karger AG, Basel.

  9. HazMatID (trademark) Replacement Project

    DTIC Science & Technology

    2013-05-09

    uses FTIR spectroscopy. It has the capability to identify chemical warfare agents, explosives , toxic industrial chemicals, narcotics, and...off-the-shelf, Fourier transform infrared spectroscopy ( FTIR ) instruments 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT SAR 18...replacement for the Smiths Detection HazMatIDTM on the 886H allowance standard, a search of Fourier transform infrared spectroscopy ( FTIR ) instruments was

  10. SYNERGISTIC WOOD PRESERVATIVES FOR REPLACEMENT OF CCA

    EPA Science Inventory

    The objective of this project was to evaluate the potential synergistic combinations of environmentally-safe biocides as wood preservatives. These wood preservatives could be potential replacements for the heavy-metal based CCA.

    Didecyldimethylammonium chloride [DDAC] was...

  11. Respiratory function after esophageal replacement in children.

    PubMed

    Gallo, Gabriele; Vrijlandt, Elianne J L E; Arets, Hubertus G M; Koppelman, Gerard H; Van der Zee, David C; Hulscher, Jan B F; Zwaveling, Sander

    2017-03-21

    Children born with esophageal atresia require an anastomosis between the proximal and distal esophagus. When this distance is too wide (long gap esophageal atresia, LGEA) esophageal replacement strategies have to be deployed. The aim of this study was to assess long-term respiratory morbidity and lung function after esophageal replacement with either stomach (gastric pull-up, GPU) or jejunum (jejunal interposition, JI) for LGEA. Retrospective cohort study. Patients operated with GPU and JI for LGEA (1985-2007) underwent a semi-structured interview and lung function testing (LFT). Seven GPU-patients and eight JI-patients were included. Median age was 12years. One patient per group could not perform LFT. Respiratory symptoms were reported by 13/15 patients (7/7 GPU-patients vs 6/8 JI-patients). All LFT items were lower than reference values; 6/13 patients showed restriction and 6/13 obstruction. All six GPU-patients had abnormal TLC and/or FEV1/FVC vs 3/7 after JI. Restriction was noted in 4/6 GPU-patients vs 2/7 JI-patients. After esophageal replacement for LGEA many children have impaired lung function and respiratory symptoms are common. Lung volumes seem decreased after GPU compared to JI. This may be caused by the intrathoracic stomach which may limit normal lung growth. Respiratory follow-up in adult life is important after esophageal replacement. III. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Aggression Replacement Training and Childhood Trauma

    ERIC Educational Resources Information Center

    Amendola, A. Mark; Oliver, Robert W.

    2013-01-01

    Aggression Replacement Training (ART) was developed by the late Arnold Goldstein of Syracuse University to teach positive alternatives to children and youth with emotional and behavioral problems (Glick & Gibbs, 2011; Goldstein, Glick, & Gibbs, 1998). ART provides cognitive, affective, and behavioral interventions to build competence in…

  13. Measure Guideline: Window Repair, Rehabilitation, and Replacement

    SciTech Connect

    Baker, P.

    2012-12-01

    This measure guideline provides information and guidance on rehabilitating, retrofitting, and replacing existing window assemblies in residential construction. The intent is to provide information regarding means and methods to improve the energy and comfort performance of existing wood window assemblies in a way that takes into consideration component durability, in-service operation, and long term performance of the strategies.

  14. Robotic Mitral Valve Replacement in Pectus Excavatum.

    PubMed

    Onan, Burak; Bakir, Ihsan

    2016-05-01

    Exposure of the mitral valve can be challenging using conventional sternotomy and thoracotomy incisions in patients with pectus deformity. We report the use of a robotic approach to replace a rheumatic mitral valve in a patient with pectus excavatum. doi: 10.1111/jocs.12740 (J Card Surg 2016;31:306-308). © 2016 Wiley Periodicals, Inc.

  15. Aggression Replacement Training and Childhood Trauma

    ERIC Educational Resources Information Center

    Amendola, A. Mark; Oliver, Robert W.

    2013-01-01

    Aggression Replacement Training (ART) was developed by the late Arnold Goldstein of Syracuse University to teach positive alternatives to children and youth with emotional and behavioral problems (Glick & Gibbs, 2011; Goldstein, Glick, & Gibbs, 1998). ART provides cognitive, affective, and behavioral interventions to build competence in…

  16. Mitochondrial replacement approaches: challenges for clinical implementation.

    PubMed

    Klopstock, Thomas; Klopstock, Barbara; Prokisch, Holger

    2016-11-25

    The advent of mitochondrial replacement techniques poses many scientific, regulatory, and ethical questions. Previous studies suggest good safety and efficacy profiles of these techniques, but challenges remain for clinical implementation and international consensus is needed on the regulation of these approaches.

  17. Knee disarticulation after total-knee replacement.

    PubMed

    Lambregts, S A M; Hitters, W M G C

    2002-12-01

    An 89-year-old woman who had a total-knee replacement in the past, underwent a knee disarticulation of the same leg because of an ischaemic foot. Eight (8) months postoperatively the stump is fully weight-bearing and the patient is able to walk safely, using a prosthesis and a walking frame.

  18. A Helix Replacement Mechanism Directs Metavinculin Functions

    SciTech Connect

    Rangarajan, Erumbi S.; Lee, Jun Hyuck; Yogesha, S.D.; Izard, Tina

    2010-10-11

    Cells require distinct adhesion complexes to form contacts with their neighbors or the extracellular matrix, and vinculin links these complexes to the actin cytoskeleton. Metavinculin, an isoform of vinculin that harbors a unique 68-residue insert in its tail domain, has distinct actin bundling and oligomerization properties and plays essential roles in muscle development and homeostasis. Moreover, patients with sporadic or familial mutations in the metavinculin-specific insert invariably develop fatal cardiomyopathies. Here we report the high resolution crystal structure of the metavinculin tail domain, as well as the crystal structures of full-length human native metavinculin (1,134 residues) and of the full-length cardiomyopathy-associated {Delta}Leu954 metavinculin deletion mutant. These structures reveal that an {alpha}-helix (H1{prime}) and extended coil of the metavinculin insert replace {alpha}-helix H1 and its preceding extended coil found in the N-terminal region of the vinculin tail domain to form a new five-helix bundle tail domain. Further, biochemical analyses demonstrate that this helix replacement directs the distinct actin bundling and oligomerization properties of metavinculin. Finally, the cardiomyopathy associated {Delta}Leu954 and Arg975Trp metavinculin mutants reside on the replaced extended coil and the H1{prime} {alpha}-helix, respectively. Thus, a helix replacement mechanism directs metavinculin's unique functions.

  19. Steam-generator replacement sets new marks

    SciTech Connect

    Beck, R.L.

    1995-04-01

    This article describes how, in one of the most successful steam-generator replacement experiences at PWRs worldwide, the V C Summer retrofit exceeded plant goals for critical-path duration, radiation, exposure, and radwaste generation. Intensive planning and teamwork, combined with the firm support of station management and the use of mockups to prepare the work crews for activity in a radiological environment, were key factors in the record performance achieved by South Carolina Electric and Gas Co (SCE and G) in replacing three steam generators at V C Summer nuclear station. The 97-day, two-hour breaker-to-breaker replacement outage -- including an eight-day delay for repair of leak in a small-bore seal-injection line of a reactor coolant pump (unrelated to the replacement activities) -- surpassed the project goal by over one day. Moreover, the outage was only 13 hours shy of the world record held by Virginia Power Co`s North Anna Unit 1.

  20. Fuglesang replaces TV Camera during first EVA

    NASA Image and Video Library

    2006-12-13

    ISS014-E-09561 (12 Dec. 2006) --- European Space Agency (ESA) astronaut Christer Fuglesang, STS-116 mission specialist, replaces a faulty TV camera on the exterior of the International Space Station during the mission's first of three planned sessions of extravehicular activity (EVA). Astronaut Robert L. Curbeam, Jr. (out of frame), mission specialist, also participated in the 6-hour, 36-minute spacewalk.

  1. Mitochondrial Replacement Techniques: Divergence in Global Policy.

    PubMed

    Schandera, Johanna; Mackey, Tim K

    2016-07-01

    In 2015, the UK became the first country permitting the clinical application of mitochondrial replacement techniques (MRT). Here, we explore how MRT have led to diverging international policy. In response, we recommend focused regulatory efforts coupled with United Nations (UN) leadership to build international consensus on the future of MRT.

  2. 24 CFR 880.602 - Replacement reserve.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 4 2013-04-01 2013-04-01 false Replacement reserve. 880.602 Section 880.602 Housing and Urban Development REGULATIONS RELATING TO HOUSING AND URBAN DEVELOPMENT... AND URBAN DEVELOPMENT (SECTION 8 HOUSING ASSISTANCE PROGRAMS, SECTION 202 DIRECT LOAN PROGRAM,...

  3. Student Aggression: Prevention, Management, and Replacement Training.

    ERIC Educational Resources Information Center

    Goldstein, Arnold P.; And Others

    American society is violent, a fact which is well-reflected in schools. This book, designed specifically for school personnel, presents the primary techniques currently being employed by educators to prevent, manage, and replace student aggression. The volume opens with a description of the origins of aggressive behavior and offers some…

  4. Life cycle benefits of calcium silicate replacements

    SciTech Connect

    Cabrera, J.G.; Woolley, G.R.

    1996-12-31

    The most universal of all binders, produced in huge quantities worldwide, is Portland cement. Following introduction in 1824 by Joseph Aspin, it has quite properly enjoyed popular appeal with craftsmen, architects and engineers for its bonding properties. The manufacture of cement relies largely on primary minerals for feedstock and depends heavily on carboniferous fuels for production. Naturally occurring minerals have similar properties as a binder and have been used in their own right or as partial replacements for cement. Likewise, secondary products recovered from modern manufacturing or energy production processes also have the potential to replace in part a proportion of cement. Life cycle analysis of cement production supports the proposition that large quantities of fossil fuel and primary minerals are being unnecessarily used in situations where alternative replacements are available and frequently placed to waste. This paper considers the implications of cement production on a sustainable environment. It argues that, contrary to some opinions, partial replacement of cement with secondary wastes which have potential cementitious properties can enhance and improve structures which otherwise would include only Portland cement as the binder. Acceptance of this argument would leave massive quantities of primary minerals untouched and reduce the amount of fossil fuels demanded of cement production on the present scale.

  5. New developments in nucleus pulposus replacement technology.

    PubMed

    Carl, Allen; Ledet, Eric; Yuan, Hansen; Sharan, Alok

    2004-01-01

    Attempts to alleviate the pain attributed to degeneration of the nucleus pulposus using replacement or reinforcement techniques dating back to the 1950s are reviewed. The various materials and their insertion techniques are discussed as are results available from early clinical experiences. These techniques are in evolution and clinical outcomes will be necessary to establish the efficacy of these approaches.

  6. Analogue Assessment of the Replacement Behavior

    ERIC Educational Resources Information Center

    Cipani, Ennio

    2008-01-01

    The use of experimental analyses in determining behavioral function for problems behaviors is well established. Such analyses lead to functional treatment prescriptions for the target problem behavior. However, data indicative of the strength of the replacement behavior are often not collected during a functional behavioral assessment. I examine…

  7. 48 CFR 8.715 - Replacement commodities.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false Replacement commodities. 8.715 Section 8.715 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION ACQUISITION PLANNING REQUIRED SOURCES OF SUPPLIES AND SERVICES Acquisition From Nonprofit Agencies Employing People Who...

  8. Is physiological glucocorticoid replacement important in children?

    PubMed Central

    Porter, John; Blair, Joanne; Ross, Richard J

    2017-01-01

    Cortisol has a distinct circadian rhythm with low concentrations at night, rising in the early hours of the morning, peaking on waking and declining over the day to low concentrations in the evening. Loss of this circadian rhythm, as seen in jetlag and shift work, is associated with fatigue in the short term and diabetes and obesity in the medium to long term. Patients with adrenal insufficiency on current glucocorticoid replacement with hydrocortisone have unphysiological cortisol concentrations being low on waking and high after each dose of hydrocortisone. Patients with adrenal insufficiency complain of fatigue, a poor quality of life and there is evidence of poor health outcomes including obesity potentially related to glucocorticoid replacement. New technologies are being developed that deliver more physiological glucocorticoid replacement including hydrocortisone by subcutaneous pump, Plenadren, a once-daily modified-release hydrocortisone and Chronocort, a delayed and sustained absorption hydrocortisone formulation that replicates the overnight profile of cortisol. In this review, we summarise the evidence regarding physiological glucocorticoid replacement with a focus on relevance to paediatrics. PMID:27582458

  9. [Hormone replacement therapy: curse or blessing?].

    PubMed

    Schmidt, M; Fink, D; Lang, U; Kimmig, R

    2006-01-01

    There is a controversial discussion on the risks and benefits of hormonal replacement therapy (HRT), and many women and doctors have revised their opinions of HRT over the last few years. Complementary and alternative therapies can be considered an option to treat menopausal symptoms. The following issue summarizes the actual knowledge of treatment options of menopausal symptoms.

  10. 24 CFR 891.745 - Replacement reserve.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... mortgage amount (for all other projects), or such higher rate as required by HUD. For the purposes of this... the Elderly and Persons with Disabilities Section 202 Projects for the Nonelderly Handicapped Families... replacement reserve are provided in § 891.605. For projects funded under §§ 891.655 through 891.790, the...

  11. A replaceable reflective film for solar concentrators

    NASA Astrophysics Data System (ADS)

    1991-09-01

    The 3M Company manufactures a silvered acrylic film called ECP-305 that is regarded as the preferred reflective film for use on stretched-membrane heliostats. However, ECP-305 will degrade in time, due to both corrosion of the silver layer and delamination at the film's silver-to-acrylic interface. The purpose of this investigation was the development of a replaceable reflector, a reflective film that can be easily removed and replaced. A replaceable reflector was successfully configured by laminating ECP-305 to the top surface of a smooth, dimensionally stable polymer film, with a removable adhesive applied to the underside of the polymer film. Several stages of screening and testing led to the selection of a 0.010-inch thick polycarbonate as the best polymer film and a medium tack tape was selected as the best removable adhesive. To demonstrate the feasibility of the replaceable reflector concept and to provide a real time field test, the chosen construction was successfully applied to the 50 sq m SKI heliostat at Sandia National Laboratories in Albuquerque.

  12. ANNUAL ESTIMATED REPLACEMENT FARMER OPPORTUNITIES IN NEBRASKA.

    ERIC Educational Resources Information Center

    GENEREUX, DOUGLAS G.

    AN ESTIMATE OF ANNUAL FARMER REPLACEMENT NEEDS IN THE 93 COUNTIES OF NEBRASKA WAS COMPUTED BY A FORMULA DEVELOPED BY CLARENCE BUNDY AT IOWA STATE UNIVERSITY AND APPLIED TO CENSUS DATA. THE FORMULA WAS USED TO DETERMINE THE NUMBER OF FARMER RETIREMENTS, OR FARMERS LEAVING FARMING, AND FARM CONSOLIDATION RATES. ACCORDING TO THE ESTIMATES, 951…

  13. Mineral replacement front propagation in deformed rocks

    NASA Astrophysics Data System (ADS)

    Beaudoin, Nicolas; Koehn, Daniel; Kelka, Ulrich

    2015-04-01

    Fluid migrations are a major agent of contaminant transport leading to mineral replacement in rocks, impacting their properties as porosity, permeability, and rheology. Understanding the physical and chemical mechanisms that govern mineralogical replacement during and after deformation is required to better understand complex interplays between fluid and rocks that are involved in faulting, seismic cycle, and resource distribution in the upper crust. Dolomitization process related to hydrothermal fluid flow is one of the most studied and debated replacement processes in earth sciences. Dolomitization of limestone is of economic importance as well, as it stands as unconventional oil reservoirs and is systematically observed in Mississippian-Valley Type ore deposit. Despite recent breakthrough about dolomitization processes at large-scale, the small-scale propagation of the reaction front remains unclear. It is poorly documented in the occurrence of stylolites and fractures in the medium while pressure-solution and fracture network development are the most efficient deformation accomodation mechanism in limestone from early compaction to layer-parallel shortening. Thus, the impact of such network on geometry of replaced bodies and on replacement front propagation deserves specific attention. This contribution illustrates the role of fracture and stylolites on the propagation of a reaction front. In a 2 dimensional numerical model we simulate the dolomitization front propagation in a heterogeneous porous medium. The propagation of the reaction front is governed by the competition between advection and diffusion processes, and takes into account reaction rates, disorder in the location of the potential replacement seeds, and permeability heterogeneities. We add stylolites and fractures that can act as barriers or drains to fluid flow according to their orientation and mineralogical content, which can or cannot react with the contaminant. The patterns produced from

  14. Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations.

    PubMed

    Kang, Eunju; Wu, Jun; Gutierrez, Nuria Marti; Koski, Amy; Tippner-Hedges, Rebecca; Agaronyan, Karen; Platero-Luengo, Aida; Martinez-Redondo, Paloma; Ma, Hong; Lee, Yeonmi; Hayama, Tomonari; Van Dyken, Crystal; Wang, Xinjian; Luo, Shiyu; Ahmed, Riffat; Li, Ying; Ji, Dongmei; Kayali, Refik; Cinnioglu, Cengiz; Olson, Susan; Jensen, Jeffrey; Battaglia, David; Lee, David; Wu, Diana; Huang, Taosheng; Wolf, Don P; Temiakov, Dmitry; Belmonte, Juan Carlos Izpisua; Amato, Paula; Mitalipov, Shoukhrat

    2016-12-08

    Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children, with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue. Pathogenic mtDNA mutations are relatively common, with an estimated 778 affected children born each year in the United States. Mitochondrial replacement therapies or techniques (MRT) circumventing mother-to-child mtDNA disease transmission involve replacement of oocyte maternal mtDNA. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother's oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer, resulting in embryos containing >99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However, some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor-to-maternal mtDNA interactions, it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition, some haplotypes confer proliferative and growth advantages to cells. Hence, we propose a matching paradigm for selecting compatible donor mtDNA for MRT.

  15. Influence of Total Knee Arthroplasty on Gait Mechanics of the Replaced and Non-Replaced Limb During Stair Negotiation.

    PubMed

    Standifird, Tyler W; Saxton, Arnold M; Coe, Dawn P; Cates, Harold E; Reinbolt, Jeffrey A; Zhang, Songning

    2016-01-01

    This study compared biomechanics during stair ascent in replaced and non-replaced limbs of total knee arthroplasty (TKA) patients with control limbs of healthy participants. Thirteen TKA patients and fifteen controls performed stair ascent. Replaced and non-replaced knees of TKA patients were less flexed at contact compared to controls. The loading response peak knee extension moment was greater in control and non-replaced knees compared with replaced. The push-off peak knee abduction moment was elevated in replaced limbs compared to controls. Loading and push-off peak hip abduction moments were greater in replaced limbs compared to controls. The push-off peak hip abduction moment was greater in non-replaced limbs compared to controls. Future rehabilitation protocols should consider the replaced knee and also the non-replaced knee and surrounding joints.

  16. Blood conservation strategies in cardiac valve replacement

    PubMed Central

    Zheng, Junnan; Chen, Liangwei; Qian, Linfeng; Jiang, Jianjie; Chen, Yinglian; Xie, Jue; Shi, Liping; Ni, Yiming; Zhao, Haige

    2016-01-01

    Abstract We aimed to evaluate whether blood conservation strategies including intraoperative autologous donation (IAD) could reduce perioperative blood transfusion for patients undergoing cardiac valve replacement including mitral valve replacement, aortic valve replacement (AVR), and double valve replacement (DVR). A total of 726 patients were studied over a 3-year period (2011–2013) after the implementation of IAD and were compared with 919 patients during the previous 36-month period (January 2008–December 2010). The method of small-volume retrograde autologous priming, strict blood transfusion standard together with IAD constituted a progressive blood-saving strategy. Baseline characteristics and preoperative information showed no statistically significant difference between IAD group and non-IAD group. Most of the postoperative morbidities are statistically the same in the 2 groups. Chest tube output (415.2 vs 1029.8 mL, P < 0.001) and postoperative respiratory failure (5.9% vs 8.6%, P = 0.039) favored the IAD group, whereas hematocrit levels were more favorable in the non-IAD group (30.3% vs 33.0% at the end of the operation, P < 0.001; 30.4% vs 31.5% at the time of discharge). The use of blood product transfusion was higher in the non-IAD group (22.6% vs 43.3%, P < 0.001). Binary multivariate logistic regression analysis showed that high age, non-IAD, DVR surgery, and absent smoking history are associated with a higher risk of intra-/postoperative blood transfusion. Blood conservation is effective and safe in cardiac valve replacement surgeries. The use of intraoperative autologous donation can lead to improved outcomes including a significantly lower rate of intra-/postoperative blood transfusion and postoperative complications. PMID:27741149

  17. Coronary Ostial Stenosis after Aortic Valve Replacement

    PubMed Central

    Ziakas, Antonios G.; Economou, Fotios I.; Charokopos, Nicholas A.; Pitsis, Antonios A.; Parharidou, Despina G.; Papadopoulos, Thomas I.; Parharidis, Georgios E.

    2010-01-01

    Coronary ostial stenosis is a rare but potentially serious sequela after aortic valve replacement. It occurs in the left main or right coronary artery after 1% to 5% of aortic valve replacement procedures. The clinical symptoms are usually severe and may appear from 1 to 6 months postoperatively. Although the typical treatment is coronary artery bypass grafting, patients have been successfully treated by means of percutaneous coronary intervention. Herein, we present the cases of 2 patients in whom coronary ostial stenosis developed after aortic valve replacement. In the 1st case, a 72-year-old man underwent aortic valve replacement and bypass grafting of the saphenous vein to the left anterior descending coronary artery. Six months later, he experienced a non-ST-segment-elevation myocardial infarction. Coronary angiography revealed a critical stenosis of the right coronary artery ostium. In the 2nd case, a 78-year-old woman underwent aortic valve replacement and grafting of the saphenous vein to an occluded right coronary artery. Four months later, she experienced unstable angina. Coronary angiography showed a critical left main coronary artery ostial stenosis and occlusion of the right coronary artery venous graft. In each patient, we performed percutaneous coronary intervention and deployed a drug-eluting stent. Both patients were asymptomatic on 6-to 12-month follow-up. We attribute the coronary ostial stenosis to the selective ostial administration of cardioplegic solution during surgery. We conclude that retrograde administration of cardioplegic solution through the coronary sinus may reduce the incidence of postoperative coronary ostial stenosis, and that stenting may be an efficient treatment option. PMID:20844624

  18. 48 CFR 908.7101-4 - Replacement of motor vehicles.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false Replacement of motor....7101-4 Replacement of motor vehicles. (a) The replacement of motor vehicles shall be in accordance with... Activities may arrange to sell, as exchange sales, used motor vehicles being replaced and to apply the...

  19. 25 CFR 700.183 - Determination of replacement housing benefit.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 2 2010-04-01 2010-04-01 false Determination of replacement housing benefit. 700.183... RELOCATION PROCEDURES Replacement Housing Payments § 700.183 Determination of replacement housing benefit. (a) Amount of benefit. The replacement housing benefit for a certified eligible head-of-household is...

  20. 25 CFR 700.189 - Expenditure of replacement home benefits.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 25 Indians 2 2012-04-01 2012-04-01 false Expenditure of replacement home benefits. 700.189 Section 700.189 Indians THE OFFICE OF NAVAJO AND HOPI INDIAN RELOCATION COMMISSION OPERATIONS AND RELOCATION PROCEDURES Replacement Housing Payments § 700.189 Expenditure of replacement home benefits. Replacement home...

  1. 25 CFR 700.189 - Expenditure of replacement home benefits.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 25 Indians 2 2014-04-01 2014-04-01 false Expenditure of replacement home benefits. 700.189 Section 700.189 Indians THE OFFICE OF NAVAJO AND HOPI INDIAN RELOCATION COMMISSION OPERATIONS AND RELOCATION PROCEDURES Replacement Housing Payments § 700.189 Expenditure of replacement home benefits. Replacement home...

  2. 25 CFR 700.189 - Expenditure of replacement home benefits.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 25 Indians 2 2013-04-01 2013-04-01 false Expenditure of replacement home benefits. 700.189 Section 700.189 Indians THE OFFICE OF NAVAJO AND HOPI INDIAN RELOCATION COMMISSION OPERATIONS AND RELOCATION PROCEDURES Replacement Housing Payments § 700.189 Expenditure of replacement home benefits. Replacement home...

  3. 25 CFR 700.189 - Expenditure of replacement home benefits.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 25 Indians 2 2011-04-01 2011-04-01 false Expenditure of replacement home benefits. 700.189 Section 700.189 Indians THE OFFICE OF NAVAJO AND HOPI INDIAN RELOCATION COMMISSION OPERATIONS AND RELOCATION PROCEDURES Replacement Housing Payments § 700.189 Expenditure of replacement home benefits. Replacement home...

  4. 25 CFR 700.189 - Expenditure of replacement home benefits.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 2 2010-04-01 2010-04-01 false Expenditure of replacement home benefits. 700.189 Section 700.189 Indians THE OFFICE OF NAVAJO AND HOPI INDIAN RELOCATION COMMISSION OPERATIONS AND RELOCATION PROCEDURES Replacement Housing Payments § 700.189 Expenditure of replacement home benefits. Replacement...

  5. Human acylphosphatase cannot replace phosphoglycerate kinase in Saccharomyces cerevisiae.

    PubMed

    Van Hoek, P; Modesti, A; Ramponi, G; Kötter, P; van Dijken, J P; Pron, J T

    2001-10-01

    Human acylphosphatase (h-AP, EC 3.6.1.7) has been reported to catalyse the hydrolysis of the 1-phosphate group of 1,3-diphosphoglycerate. In vivo operation of this reaction in the yeast Saccharomyces cerevisiae would bypass phosphoglycerate kinase and thus reduce the ATP yield from glycolysis. To investigate whether h-AP can indeed replace the S. cerevisiae phosphoglycerate kinase, a multi-copy plasmid carrying the h-AP gene under control of the yeast TDH3 promoter was introduced into a pgk1 delta mutant of S. cerevisiae. A strain carrying the expression vector without the h-AP cassette was used as a reference. For both strains, steady-state carbon- and energy-limited chemostat cultures were obtained at a dilution rate of 0.10 h(-1) on a medium containing a mixture of glucose and ethanol (15% and 85% on a carbon basis, respectively). Although the h-AP strain exhibited a high acylphosphatase activity in cell extracts, switching to glucose as sole carbon and energy source resulted in a complete arrest of glucose consumption and growth. The lack of a functional glycolytic pathway was further evident from the absence of ethanol formation in the presence of excess glucose in the culture. As h-AP cannot replace yeast phosphoglycerate kinase in vivo, the enzyme is not a useful tool to modify the ATP yield of glycolysis in S. cerevisiae.

  6. Seamless stitching of biosynthetic gene cluster containing type I polyketide synthases using Red/ET mediated recombination for construction of stably co-existing plasmids.

    PubMed

    Su, Chun; Zhao, Xin-Qing; Wang, Hai-Na; Qiu, Rong-Guo; Tang, Li

    2015-01-10

    Type I polyketides are natural products with diverse functions that are important for medical and agricultural applications. Manipulation of large biosynthetic gene clusters containing type I polyketide synthases (PKS) for heterologous expression is difficult due to the existence of conservative sequences of PKS in multiple modules. Red/ET mediated recombination has permitted rapid manipulation of large fragments; however, it requires insertion of antibiotic selection marker in the cassette, raising the problem of interference of expression by leaving "scar" sequence. Here, we report a method for precise seamless stitching of large polyketide biosynthetic gene cluster using a 48.4kb fragment containing type I PKS involved in fostriecin biosynthesis as an example. rpsL counter-selection was used to assist seamless stitching of large fragments, where we have overcome both the size limitations and the restriction on endonuclease sites during the Red/ET recombination. The compatibility and stability of the co-existing vectors (p184 and pMT) which respectively accommodate 16kb and 32.4kb inserted fragments were demonstrated. The procedure described here is efficient for manipulation of large DNA fragments for heterologous expression. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. A methodology for markerless genetic modifications in Azotobacter vinelandii.

    PubMed

    Eberhart, L J; Knutson, C M; Barney, B M

    2016-06-01

    Efficient manipulation of multiple regions within a genome can be improved by counter-selection approaches. In this work, we sought to develop a method to manipulate Azotobacter vinelandii using a counter-selection approach based on the presence of the pyrF gene. A background uracil auxotroph of A. vinelandii was first constructed by deleting the pyrF gene coding orotidine-5'-phosphate decarboxylase. The pyrF gene and promoter were also incorporated together with an antibiotic marker to create a selection and counter-selection cassette to shuttle into various plasmids. The constructed cassette could then be removed using a plasmid lacking the pyrF gene via counter-selection resulting from the production of 5-fluorouracil. The process could be repeated multiple times using the same procedure for selection and counter-selection. Following completion, the pyrF gene may be reintroduced to the genome in its original location, leaving a completed strain devoid of any antibiotic markers. Utilization of the pyrF gene for counter-selection is a powerful tool that can be used effectively to make multiple gene deletions in A. vinelandii. This study demonstrates the successful application of a counter-selection approach to yield markerless genetic modifications to A. vinelandii, which should be of interest for a range of applications in this important model bacterium. © 2016 The Society for Applied Microbiology.

  8. Novel synthetic Medea selfish genetic elements drive population replacement in Drosophila; a theoretical exploration of Medea-dependent population suppression.

    PubMed

    Akbari, Omar S; Chen, Chun-Hong; Marshall, John M; Huang, Haixia; Antoshechkin, Igor; Hay, Bruce A

    2014-12-19

    Insects act as vectors for diseases of plants, animals, and humans. Replacement of wild insect populations with genetically modified individuals unable to transmit disease provides a potentially self-perpetuating method of disease prevention. Population replacement requires a gene drive mechanism in order to spread linked genes mediating disease refractoriness through wild populations. We previously reported the creation of synthetic Medea selfish genetic elements able to drive population replacement in Drosophila. These elements use microRNA-mediated silencing of myd88, a maternally expressed gene required for embryonic dorso-ventral pattern formation, coupled with early zygotic expression of a rescuing transgene, to bring about gene drive. Medea elements that work through additional mechanisms are needed in order to be able to carry out cycles of population replacement and/or remove existing transgenes from the population, using second-generation elements that spread while driving first-generation elements out of the population. Here we report the synthesis and population genetic behavior of two new synthetic Medea elements that drive population replacement through manipulation of signaling pathways involved in cellular blastoderm formation or Notch signaling, demonstrating that in Drosophila Medea elements can be generated through manipulation of diverse signaling pathways. We also describe the mRNA and small RNA changes in ovaries and early embryos associated from Medea-bearing females. Finally, we use modeling to illustrate how Medea elements carrying genes that result in diapause-dependent female lethality could be used to bring about population suppression.

  9. H3.3 replacement facilitates epigenetic reprogramming of donor nuclei in somatic cell nuclear transfer embryos.

    PubMed

    Wen, Duancheng; Banaszynski, Laura A; Rosenwaks, Zev; Allis, C David; Rafii, Shahin

    2014-01-01

    Transfer of a somatic nucleus into an enucleated oocyte is the most efficient approach for somatic cell reprogramming. While this process is known to involve extensive chromatin remodeling of the donor nucleus, the maternal factors responsible and the underlying chromatin-based mechanisms remain largely unknown. Here we discuss our recent findings demonstrating that the histone variant H3.3 plays an essential role in reprogramming and is required for reactivation of key pluripotency genes in somatic cell nuclear transfer (SCNT) embryos. Maternal-derived H3.3 replaces H3 in the donor nucleus shortly after oocyte activation, with the amount of replacement directly related to the differentiation status of the donor nucleus in SCNT embryos. We provide additional evidence to suggest that de novo synthesized H3.3 replaces histone H3 carrying repressive modifications in the donor nuclei of SCNT embryos, and hypothesize that replacement may occur at specific loci that must be reprogrammed for gene reactivation.

  10. The road we travelled: discovery, choreography, and significance of brain replaceable neurons.

    PubMed

    Nottebohm, Fernando

    2004-06-01

    Neurons are constantly added to the telencephalon of songbirds. In the high vocal center (HVC), where this has been studied, new neurons replace older ones that died. Peaks in replacement are seasonal and affect some neuronal classes but not others. Peaks in replacement coincide with peaks in information acquisition. The new neurons are produced by division of cells in the wall of the lateral ventricle. Where studied closely, the neuronal stem cells proved to be radial glia. Life expectancy of the new neurons ranges from weeks to months. New neuron survival is regulated by vacancies, hormones, and activity. The immediate agent of new neuron survival is, in some cases, brain-derived neurotrophic factor (BDNF). The effect of BDNF is maximal 14-20 days after the cells are born, when they are establishing their connections. These observations are now being extended to other vertebrates and may apply, to varying degrees, to all of them. The function of neuronal replacement in healthy adult brain remains unclear. If synaptic number and efficacy sufficed as mechanisms for long-term memory storage and could be adjusted again and again to incorporate new memories, then neuronal replacement would seem unnecessary. Since it occurs, it seems reasonable to suppose that replacement serves to maintain learning potential in a way that could not be done just by synaptic change. Long-term memories may be encoded by long-term changes in gene expression akin to a last step in cell differentiation. If so, neuronal replacement may be the adult brain's way of striking a balance between limited memory space and the need to acquire new memories. The testing of this hypothesis remains in the future. This chapter tells how neuronal replacement was discovered in the adult songbird brain.

  11. A complex method of equipment replacement planning. An advanced plan for the replacement of medical equipment.

    PubMed

    Dondelinger, Robert M

    2004-01-01

    This complex method of equipment replacement planning is a methodology; it is a means to an end, a process that focuses on equipment most in need of replacement, rather than the end itself. It uses data available from the maintenance management database, and attempts to quantify those subjective items important [figure: see text] in making equipment replacement decisions. Like the simple method of the last issue, it is a starting point--albeit an advanced starting point--which the user can modify to fit their particular organization, but the complex method leaves room for expansion. It is based on sound logic, documented facts, and is fully defensible during the decision-making process and will serve your organization well as provide a structure for your equipment replacement planning decisions.

  12. [Aortic valve replacement and hemiarch replacement in a patient with a porcelain aorta].

    PubMed

    Okamoto, Y; Sakakibara, K; Inoue, H; Suzuki, S; Shindo, S; Matsumoto, M

    2009-07-01

    We report a case of aortic valve replacement and hemiarch replacement with reconstruction of the brachiocephalic artery in a patient with a porcelain aorta. A 65-year-old man was admitted to the hospital for aortic stenosis. Computed tomography demonstrated severe aortic calcification and extensive calcification covering a wide area of the aorta. The echocardiography showed a highly calcified aortic valve and a pressure gradient of 109 mmHg across the aortic valve. At surgery, calcification of the ascending aorta was severe and involved its entire circumference. Therefore, 2 cannulae were inserted to the right axillary and right femoral arteries for extracorporeal circulation. We performed replacement of the ascending aorta and hemiarch with reconstruction of the brachiocephalic artery under circulatory arrest and antegrade cerebral perfusion, as well as aortic valve replacement with a 21 mm St. Jude Medical mechanical valve. He had no major cardiac complications during the postoperative course, and was discharged on postoperative day 33.

  13. Replacements For Ozone-Depleting Foaming Agents

    NASA Technical Reports Server (NTRS)

    Blevins, Elana; Sharpe, Jon B.

    1995-01-01

    Fluorinated ethers used in place of chlorofluorocarbons and hydrochlorofluorocarbons. Replacement necessary because CFC's and HCFC's found to contribute to depletion of ozone from upper atmosphere, and manufacture and use of them by law phased out in near future. Two fluorinated ethers do not have ozone-depletion potential and used in existing foam-producing equipment, designed to handle liquid blowing agents soluble in chemical ingredients that mixed to make foam. Any polyurethane-based foams and several cellular plastics blown with these fluorinated ethers used in processes as diverse as small batch pours, large sprays, or double-band lamination to make insulation for private homes, commercial buildings, shipping containers, and storage tanks. Fluorinated ethers proved useful as replacements for CFC refrigerants and solvents.

  14. Replacement Power Facility site selection report

    SciTech Connect

    Wike, L.D.; Toole, G.L.; Specht, W.L.

    1992-06-01

    The Department of Energy (DOE) has proposed the construction and operation of a Replacement Power Facility (RPF) for supplementing and replacing existing sources of steam and possibly electricity at the Savannah River Site (SRS). DOE is preparing an Environmental Impact Statement (EIS) for this project As part of the impact analysis of the proposed action, the EIS will include a detailed description of the environment where the RPF will be constructed. This description must be specific to the recommended site at SRS, which contains more than 300 square miles of land including streams, lakes, impoundments, wetlands, and upland areas. A formal site-selection process was designed and implemented to identify the preferred RPF site.

  15. Replacements For Ozone-Depleting Foaming Agents

    NASA Technical Reports Server (NTRS)

    Blevins, Elana; Sharpe, Jon B.

    1995-01-01

    Fluorinated ethers used in place of chlorofluorocarbons and hydrochlorofluorocarbons. Replacement necessary because CFC's and HCFC's found to contribute to depletion of ozone from upper atmosphere, and manufacture and use of them by law phased out in near future. Two fluorinated ethers do not have ozone-depletion potential and used in existing foam-producing equipment, designed to handle liquid blowing agents soluble in chemical ingredients that mixed to make foam. Any polyurethane-based foams and several cellular plastics blown with these fluorinated ethers used in processes as diverse as small batch pours, large sprays, or double-band lamination to make insulation for private homes, commercial buildings, shipping containers, and storage tanks. Fluorinated ethers proved useful as replacements for CFC refrigerants and solvents.

  16. Liver replacement for alpha1-antitrypsin deficiency

    PubMed Central

    Putnam, Charles W.; Porter, Kendrick A.; Peters, Robert L.; Ashcavai, Mary; Redeker, Allan G.; Starzl, Thomas E.

    2010-01-01

    A 16-year-old girl with advanced cirrhosis and severe alpha1-antitrypsin deficiency of the homozygous PiZZ phenotype was treated by orthotopic liver transplantation. After replacement of the liver with a homograft from a donor with the normal PiMM phenotype, the alpha1-antitrypsin concentration in the recipient’s serum rose to normal; it had the PiMM phenotype. Two and a third years later, chronic rejection necessitated retransplantation. Insertion of a homograft from a heterozygous PiMZ donar was followed by the identification of that phenotype in the recipient’s serum. Neither liver graft developed the alpha1-antitrypsin glycoprotein deposits seen with the deficiency state. These observations confirm that this hepatic- based inborn error metabolism is metabolically cured by liver replacement. PMID:320694

  17. Enzyme replacement therapy: conception, chaos and culmination.

    PubMed Central

    Brady, Roscoe O

    2003-01-01

    Soon after the enzymatic defects in Gaucher disease and in Niemann-Pick disease were discovered, enzyme replacement or enzyme supplementation was proposed as specific treatment for patients with these and related metabolic storage disorders. While relatively straightforward in concept, successful implementation of this approach required many years of intensive effort to bring it to fruition. Procedures were eventually developed to produce sufficient quantities of the requisite enzymes for clinical trials and to target therapeutic enzymes to lipid-storing cells. These achievements led to the development of effective enzyme replacement therapy for patients with Gaucher disease and for Fabry disease. These demonstrations provide strong incentive for the application of this strategy for the treatment of many human disorders of metabolism. PMID:12803925

  18. Heart Valve Replacements with Regenerative Capacity

    PubMed Central

    Dijkman, Petra E.; Fioretta, Emanuela S.; Frese, Laura; Pasqualini, Francesco S.; Hoerstrup, Simon P.

    2016-01-01

    The incidence of severe valvular dysfunctions (e.g., stenosis and insufficiency) is increasing, leading to over 300,000 valves implanted worldwide yearly. Clinically used heart valve replacements lack the capacity to grow, inherently requiring repetitive and high-risk surgical interventions during childhood. The aim of this review is to present how different tissue engineering strategies can overcome these limitations, providing innovative valve replacements that proved to be able to integrate and remodel in pre-clinical experiments and to have promising results in clinical studies. Upon description of the different types of heart valve tissue engineering (e.g., in vitro, in situ, in vivo, and the pre-seeding approach) we focus on the clinical translation of this technology. In particular, we will deepen the many technical, clinical, and regulatory aspects that need to be solved to endure the clinical adaptation and the commercialization of these promising regenerative valves. PMID:27721704

  19. Biosecurity and risk management for dairy replacements.

    PubMed

    Maunsell, Fiona; Donovan, G Arthur

    2008-03-01

    Biosecurity, biocontainment, and disease risk management on dairy replacement operations are time- and labor-intensive, planned programs. Oftentimes the value of these programs is realized only after disease is introduced to a facility or a disease outbreak occurs. There is no "one-plan-fits-all;" each plan must be tailored to meet the needs of management's goals and expectations and problems specific to a production enterprise or geographic region. A standard framework applicable to biosecurity programs includes: (1) hazard identification, (2) exposure assessment, (3) risk characterization, and (4) risk management. The discussion presented here helps lay the framework for development and implementation of biosecurity and risk-management programs within dairy replacement facilities.

  20. Material considerations for intervertebral disc replacement implants.

    PubMed

    Taksali, Sudeep; Grauer, Jonathan N; Vaccaro, Alexander R

    2004-01-01

    Cervical and lumbar disc replacements are being performed with increasing frequency. Much of the background for the development for these implants is drawn from the literature of other joint replacements that have been in evolution and use for decades. Important variables for the function and longevity of such disc arthroplasty implants are clearly defined by the material properties of the components used for their production. The most frequently considered materials are cobalt-chrome alloys, titanium alloys, stainless steels, polyethylene, polyurethane and ceramics. In addition to implant materials, the interfaces of such materials must be considered. The bearing surfaces of an implant, in particular, are at risk of wear and failure. Overall, successful, long-term total disc arthroplasty requires a thorough understanding of biomaterials and how they can be used to achieve their desired goals.