Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases.

Science.gov (United States)

Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

2015-10-16

Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9), hold tremendous potential for applications in the clinical setting to treat genetic diseases or prevent infectious diseases. However, because the accuracy of DNA recognition by these nucleases is not always perfect, off-target mutagenesis may result in undesirable adverse events in treated patients such as cellular toxicity or tumorigenesis. Therefore, designing nucleases and analyzing their activity must be carefully evaluated to minimize off-target mutagenesis. Furthermore, rigorous genomic testing will be important to ensure the integrity of nuclease modified cells. In this review, we provide an overview of available nuclease designing platforms, nuclease engineering approaches to minimize off-target activity, and methods to evaluate both on- and off-target cleavage of CRISPR-Cas9.

Engineering nucleases for gene targeting: safety and regulatory considerations.

Science.gov (United States)

Pauwels, Katia; Podevin, Nancy; Breyer, Didier; Carroll, Dana; Herman, Philippe

2014-01-25

Nuclease-based gene targeting (NBGT) represents a significant breakthrough in targeted genome editing since it is applicable from single-celled protozoa to human, including several species of economic importance. Along with the fast progress in NBGT and the increasing availability of customized nucleases, more data are available about off-target effects associated with the use of this approach. We discuss how NBGT may offer a new perspective for genetic modification, we address some aspects crucial for a safety improvement of the corresponding techniques and we also briefly relate the use of NBGT applications and products to the regulatory oversight. Copyright © 2013 Elsevier B.V. All rights reserved.

Both base excision repair and nucleotide excision repair in humans are influenced by nutritional factors.

Science.gov (United States)

Brevik, Asgeir; Karlsen, Anette; Azqueta, Amaya; Tirado, Anna Estaban; Blomhoff, Rune; Collins, Andrew

2011-01-01

Lack of reliable assays for DNA repair has largely prevented measurements of DNA repair from being included in human biomonitoring studies. Using newly developed modifications of the comet assay we tested whether a fruit- and antioxidant-rich plant-based intervention could affect base excision repair (BER) and nucleotide excision repair (NER) in a group of 102 male volunteers. BER and NER repair capacities were measured in lymphocytes before and after a dietary intervention lasting 8 weeks. The study had one control group, one group consuming three kiwifruits per day and one group consuming a variety of antioxidant-rich fruits and plant products in addition to their normal diet. DNA strand breaks were reduced following consumption of both kiwifruits (13%, p = 0.05) and antioxidant-rich plant products (20%, p = 0.02). Increased BER (55%, p = 0.01) and reduced NER (-39%, p plant products. Reduced NER was also observed in the kiwifruit group (-38%, p = 0.05), but BER was not affected in this group. Here we have demonstrated that DNA repair is affected by diet and that modified versions of the comet assay can be used to assess activity of different DNA repair pathways in human biomonitoring studies. Copyright © 2010 John Wiley & Sons, Ltd.

Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

Science.gov (United States)

Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2017-10-01

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once.

Science.gov (United States)

Tsutakawa, Susan E; Lafrance-Vanasse, Julien; Tainer, John A

2014-07-01

To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by James Watson and Francis Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure

Origins of Programmable Nucleases for Genome Engineering.

Science.gov (United States)

Chandrasegaran, Srinivasan; Carroll, Dana

2016-02-27

Genome engineering with programmable nucleases depends on cellular responses to a targeted double-strand break (DSB). The first truly targetable reagents were the zinc finger nucleases (ZFNs) showing that arbitrary DNA sequences could be addressed for cleavage by protein engineering, ushering in the breakthrough in genome manipulation. ZFNs resulted from basic research on zinc finger proteins and the FokI restriction enzyme (which revealed a bipartite structure with a separable DNA-binding domain and a non-specific cleavage domain). Studies on the mechanism of cleavage by 3-finger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target sequence recognition from 9 to 18bp, long enough to specify a unique genomic locus in plant and mammalian cells. Soon afterwards, a ZFN-induced DSB was shown to stimulate homologous recombination in cells. Transcription activator-like effector nucleases (TALENs) that are based on bacterial TALEs fused to the FokI cleavage domain expanded this capability. The fact that ZFNs and TALENs have been used for genome modification of more than 40 different organisms and cell types attests to the success of protein engineering. The most recent technology platform for delivering a targeted DSB to cellular genomes is that of the RNA-guided nucleases, which are based on the naturally occurring Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system-the ease of RNA design for new targets and the dependence on a single, constant Cas9 protein-have led to its wide adoption by research laboratories around the world. These technology platforms have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with wide-ranging implications across biology and medicine. However, these nucleases have also been shown to cut

Antitumor Effects and Cytotoxicity of Recombinant Plant Nucleases

Czech Academy of Sciences Publication Activity Database

Matoušek, Jaroslav; Podzimek, Tomáš; Pouckova, P.; Stehlík, Jan; Škvor, J.; Souček, J.; Matoušek, Josef

2009-01-01

Roč. 18, č. 4 (2009), s. 163-171 ISSN 0965-0407 R&D Projects: GA ČR GA521/09/1214 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z50450515 Keywords : Anticarcinogenic and antiproliferative nucleases * Human melanoma * Tumor xenografts * Nicotiana benthamina Subject RIV: FD - Oncology ; Hematology Impact factor: 1.478, year: 2009

Identification of a residue critical for the excision of 3′-blocking ends in apurinic/apyrimidinic endonucleases of the Xth family

Science.gov (United States)

Castillo-Acosta, Víctor M.; Ruiz-Pérez, Luis M.; Yang, Wei; González-Pacanowska, Dolores; Vidal, Antonio E.

2009-01-01

DNA single-strand breaks containing 3′-blocking groups are generated from attack of the sugar backbone by reactive oxygen species or after base excision by DNA glycosylase/apurinic/apyrimidinic (AP) lyases. In human cells, APE1 excises sugar fragments that block the 3′-ends thus facilitating DNA repair synthesis. In Leishmania major, the causal agent of leishmaniasis, the APE1 homolog is the class II AP endonuclease LMAP. Expression of LMAP but not of APE1 reverts the hypersensitivity of a xth nfo repair-deficient Escherichia coli strain to the oxidative compound hydrogen peroxide (H2O2). To identify the residues specifically involved in the repair of oxidative DNA damage, we generated random mutations in the ape1 gene and selected those variants that conferred protection against H2O2. Among the resistant clones, we isolated a mutant in the nuclease domain of APE1 (D70A) with an increased capacity to remove 3′-blocking ends in vitro. D70 of APE1 aligns with A138 of LMAP and mutation of the latter to aspartate significantly reduces its 3′-phosphodiesterase activity. Kinetic analysis shows a novel role of residue D70 in the excision rate of 3′-blocking ends. The functional and structural differences between the parasite and human enzymes probably reflect a divergent molecular evolution of their DNA repair responses to oxidative damage. PMID:19181704

Gamma-ray excision repair in normal and diseased human cells

International Nuclear Information System (INIS)

Cerutti, P.A.; Remsen, J.F.

1976-01-01

Radiation products of the 5,6-dihydroxy-dihydrothymine type (t') are efficiently removed from the DNA during postirradiation incubation of bacterial and mammalian cells. In this chapter we describe the t'-excision system contained in normal human cells, in human carcinoma HeLa S-3 cells, and in skin fibroblasts from xeroderma pigmentosum (XP) and Fanconi's anemia (FA) patients. The latter diseases are characterized among other symptoms by a genetically increased susceptibility for the development of cancer

Structural characterization of the virulence factor nuclease A from Streptococcus agalactiae.

Science.gov (United States)

Moon, Andrea F; Gaudu, Philippe; Pedersen, Lars C

2014-11-01

The group B pathogen Streptococcus agalactiae commonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Nuclease A (GBS_NucA), a secreted DNA/RNA nuclease, serves as a virulence factor for S. agalactiae, facilitating bacterial evasion of the human innate immune response. GBS_NucA efficiently degrades the DNA matrix component of neutrophil extracellular traps (NETs), which attempt to kill and clear invading bacteria during the early stages of infection. In order to better understand the mechanisms of DNA substrate binding and catalysis of GBS_NucA, the high-resolution structure of a catalytically inactive mutant (H148G) was solved by X-ray crystallography. Several mutants on the surface of GBS_NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique. While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity. These structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS_NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted to S. agalactiae.

Differing levels of excision repair in human fetal dermis and brain cells

International Nuclear Information System (INIS)

Gibson, R.E.; D'Ambrosio, S.M.; Ohio State Univ., Columbus

1982-01-01

The levels of DNA excision repair, as measured by unscheduled DNA synthesis (UDS) and the UV-endonuclease sensitive site assay, were compared in cells derived from human fetal brain and dermal tissues. The level of UDS induced following ultraviolet (UV) irradiation was found to be lower (approx. 60%) in the fetal brain cells than in fetal dermal cells. It was determined, using the UV-endonuclease sensitive site assay to confirm the UDS observation, that 50% of the dimers induced by UV in fetal dermal cells were repaired in 8 h. while only 15% were removed in the fetal brain cells during the same period of time. Even after 24 h. only 44% of the dimers induced by UV in the fetal brain cells were repaired, while 65% were removed in the dermal cells. These data suggest that cultured human fetal brain cells exhibit lower levels of excision repair compared to cultured human fetal dermal cells. (author)

Probing chromatin structure with nuclease sensitivity assays.

Science.gov (United States)

Gregory, R I; Khosla, S; Feil, R

2001-01-01

To further our understanding of genomic imprinting it will be essential to identify key control elements, and to investigate their regulation by both epigenetic modifications (such as DNA methylation) and trans-acting factors. So far, sequence elements that regulate parental allele-specific gene expression have been identified in a number of imprinted loci, either because of their differential DNA methylation or through functional studies in transgenic mice (1,2). A systematic search for allele-specific chromatin features constitutes an alternative strategy to identify elements that regulate imprinting. The validity of such an in vivo chromatin approach derives from the fact that in several known imprinting control-elements, a specialized organization of chromatin characterized by nuclease hypersensitivity is present on only one of the two parental chromosome (3). For example, the differentially methylated 5 -portion of the human SNRPN gene-a sequence element that controls imprinting in the Prader-Willi and Angelman syndromes' domain on chromosome 15q11- q13-has strong DNase-I hypersensitive sites on the unmethylated paternal chromosome (4). A differentially methylated region that regulates the imprinting of H19 and that of the neighboring insulin-like growth factor-2 gene on mouse chromosome 7 was also found to have parental chromosome-specific hypersensitive sites (5,6). The precise nature of the allelic nuclease hypersensitivity in these and other imprinted loci remains to be determined in more detail, for example, by applying complementary chromatin methodologies (7,8). However, it is commonly observed that a nuclease hypersensitive site corresponds to a small region where nucleosomes are absent or partially disrupted.

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

Science.gov (United States)

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

2015-02-18

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. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Recognition and repair of the CC-1065-(N3-Adenine)-DNA adduct by the UVRABC nuclease

International Nuclear Information System (INIS)

Tang, M.; Lee, C.S.; Doisy, R.; Ross, L.; Needham-VanDevanter, D.R.; Hurley, L.H.

1988-01-01

The recognition and repair of the helix-stabilizing and relatively nondistortive CC-1065-(N3-adenine)-DNA adduct by UVRABC nuclease has been investigated both in vivo with phi X174RFI DNA by a transfection assay and in vitro by a site-directed adduct in a 117 base pair fragment from M13mp1. CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis which binds within the minor groove of DNA through N3 of adenine. In contrast to the helix-destabilizing and distortive modifications of DNA caused by ultraviolet light or N-acetoxy-2-(acetylamino)fluorene, CC-1065 increases the melting point of DNA and decreases the S1 nuclease activity. Using a viral DNA-Escherichia coli transfection system, the authors have found that the uvrA, uvrB, and uvrC genes, which code for the major excision repair proteins for UV- and NAAAF-induced DNA damage, are also involved in the repair of CC-1065-DNA adducts. In contrast, the uvrD gene product, which has been found to be involved in the repair of UV damage, has no effect in repairing CC-1065-DNA adducts. Purified UVRA, UVRB, and UVRC proteins must work in concert to incise the drug-modified phi X174RFI DNA. Using a site-directed and multiple CC-1065 modified (MspI-BstNI) 117 base pair fragment from M13mp1, they have found that UVRABC nuclease incises at the eight phosphodiester bond on the 5' side of the CC-1065-DNA adduct on the drug-modified strand. The enzymes do not cut the noncovalently modified strand. The DNA sequence and/or helix-stabilizing effect of multiple adducts may determine the recognition and/or incision of the drug-DNA adduct by UVRABC nuclease. These results are discussed in relation to the structure of the CC-1065-DNA adduct and the effect of drug binding on local DNA structure

On pitch jumps between chest and falsetto registers in voice : Data from living and excised human larynges

NARCIS (Netherlands)

Svec, JG; Schutte, HK; Miller, DG

The paper offers a new concept of studying abrupt chest-falsetto register transitions Clumps) based on the theory of nonlinear dynamics. The jumps were studied in an excised human larynx and in three living subjects tone female and two male). Data from the excised larynx revealed that a small and

Genome engineering in human cells.

Science.gov (United States)

Song, Minjung; Kim, Young-Hoon; Kim, Jin-Soo; Kim, Hyongbum

2014-01-01

Genome editing in human cells is of great value in research, medicine, and biotechnology. Programmable nucleases including zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases recognize a specific target sequence and make a double-strand break at that site, which can result in gene disruption, gene insertion, gene correction, or chromosomal rearrangements. The target sequence complexities of these programmable nucleases are higher than 3.2 mega base pairs, the size of the haploid human genome. Here, we briefly introduce the structure of the human genome and the characteristics of each programmable nuclease, and review their applications in human cells including pluripotent stem cells. In addition, we discuss various delivery methods for nucleases, programmable nickases, and enrichment of gene-edited human cells, all of which facilitate efficient and precise genome editing in human cells.

Pyrimidine dimer excision in human cells and skin cancer

International Nuclear Information System (INIS)

Regan, J.D.; Carrier, W.L.; Smith, D.P.; Waters, R.

1977-01-01

We have compared three different methods for estimating the induction and removal of uv induced pyrimidine dimers from the DNA of human fibroblasts. Results indicate that after uv doses of 5-20 J/m 2 50% of the dimers are removed by 24 hours after irradiation. Almost complete excision can be observed if the cells are incubated for periods not less than 72 hours after 5 J/m 2 . After higher doses it probably takes even longer fr such complete removal to be seen

High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases

DEFF Research Database (Denmark)

Chen, Fuqiang; Pruett-Miller, Shondra M; Huang, Yuping

2011-01-01

Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point...

Applications of Alternative Nucleases in the Age of CRISPR/Cas9

Directory of Open Access Journals (Sweden)

Tuhin K. Guha

2017-11-01

Full Text Available Breakthroughs in the development of programmable site-specific nucleases, including zinc-finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs, meganucleases (MNs, and most recently, the clustered regularly interspaced short palindromic repeats (CRISPR associated proteins (including Cas9 have greatly enabled and accelerated genome editing. By targeting double-strand breaks to user-defined locations, the rates of DNA repair events are greatly enhanced relative to un-catalyzed events at the same sites. However, the underlying biology of each genome-editing nuclease influences the targeting potential, the spectrum of off-target cleavages, the ease-of-use, and the types of recombination events at targeted double-strand breaks. No single genome-editing nuclease is optimized for all possible applications. Here, we focus on the diversity of nuclease domains available for genome editing, highlighting biochemical properties and the potential applications that are best suited to each domain.

Transcription Activator-Like Effectors (TALEs) Hybrid Nucleases for Genome Engineering Application

KAUST Repository

Wibowo, Anjar

2011-06-06

Gene targeting is a powerful genome engineering tool that can be used for a variety of biotechnological applications. Genomic double-strand DNA breaks generated by engineered site-specific nucleases can stimulate gene targeting. Hybrid nucleases are composed of DNA binding module and DNA cleavage module. Zinc Finger Nucleases were used to generate double-strand DNA breaks but it suffers from failures and lack of reproducibility. The transcription activator–like effectors (TALEs) from plant pathogenic Xanthomonas contain a unique type of DNA-binding domain that bind specific DNA targets. The purpose of this study is to generate novel sequence specific nucleases by fusing a de novo engineered Hax3 TALE-based DNA binding domain to a FokI cleavage domain. Our data show that the de novo engineered TALE nuclease can bind to its target sequence and create double-strand DNA breaks in vitro. We also show that the de novo engineered TALE nuclease is capable of generating double-strand DNA breaks in its target sequence in vivo, when transiently expressed in Nicotiana benthamiana leaves. In conclusion, our data demonstrate that TALE-based hybrid nucleases can be tailored to bind a user-selected DNA sequence and generate site-specific genomic double-strand DNA breaks. TALE-based hybrid nucleases hold much promise as powerful molecular tools for gene targeting applications.

The involvement of nuclear nucleases in rat thymocyte DNA degradation after γ-irradiation

International Nuclear Information System (INIS)

Nikonova, L.V.; Nelipovich, P.A.; Umansky, S.R.

1982-01-01

Possible mechanisms of internucleosomal DNA fragmentation in thymocytes of irradiated rats were studied. It was shown that thymocyte nuclei contain at least two nucleases that cleave DNA between nucleosomes - a Ca 2+ /Mg 2+ -dependent nuclease and an acidic one which does not depend on bivalent ions. 2 and 3 h after irradiation at a dose of 10 Gy the initial rate of DNA cleavage by Ca 2+ /Mg 2+ -dependent nuclease in isolated nuclei increased three and seven times, respectively, but the kinetics of DNA digestion by acidic nuclease did not change. The experiments with cycloheximide indicated that Ca 2+ /Mg 2+ -dependent endonuclease turns over at a high rate. The activity of the cytoplasmic acidic and Mg 2+ -dependent nucleases was shown to increase (by 40 and 50%, respectively) 3h after irradiation. The effect is caused by the de novo synthesis of the nucleases. At the same time the activity of nuclear nucleases did not essentially change. The chromatin isolated from rat thymocytes 3 h after irradiation did not differ in its sensitivity to some exogenic nucleases (DNAase I, micrococcal nuclease and nuclease from Serratia marcescens) from the control. Thus, Ca 2+ /Mg 2+ -dependent endonuclease seems to be responsible for the postirradiation internucleosomal DNA fragmentation in dying thymocytes. (Auth.)

Intra and extracellular nuclease production by Aspergillus niger and Aspergillus nidulans

Directory of Open Access Journals (Sweden)

Ferreira Adlane V. B.

1998-01-01

Full Text Available Intra and extracellular nuclease production by strains of Aspergillus niger and Aspergillus nidulans was estimated using a modified DNAse test agar and cell-free extract assays. Differences in the production of nucleases by A. niger and A. nidulans were observed. These observations suggest that the DNAse test agar can be helpful for a quick screening for some types of nucleases in filamentous fungi. The assays using cell-free extracts can also be useful for initial characterization of other types of nucleases.

Cryopreserved Ultra-Thick Human Amniotic Membrane for Conjunctival Surface Reconstruction After Excision of Conjunctival Tumors.

Science.gov (United States)

Tanaka, Thais S; Demirci, Hakan

2016-04-01

Cryopreserved ultra-thick human amniotic membrane (AM) is used for glaucoma surgery. We evaluated the use of cryopreserved ultra-thick human AM for conjunctival surface reconstruction after excision of a conjunctival tumor. We retrospectively reviewed the medical records of 28 patients who underwent conjunctival surface reconstruction with cryopreserved ultra-thick human AM after excision of the tumor. The AM was secured to the surrounding conjunctiva and underlying sclera with interrupted 8-0 Vicryl sutures. Clinical data regarding demographics, diagnosis, size and location of conjunctival tumors, patient outcome, and complications were gathered. Of 28 patients, 6 (21.4%) had malignant melanoma, 4 (14.3%) had squamous cell carcinoma, 6 (21.4%) had conjunctival intraepithelial neoplasia, 1 (3.6%) had sebaceous carcinoma, 1 (3.6%) had mucoepidermoid carcinoma, 1 (3.6%) had conjunctival intraepithelial dysplasia, 5 (17.9%) had pterygium, 2 (7.1%) had compound nevus, 1 (3.6%) had a large epithelial inclusion cyst, and 1 (3.6%) patient had a granuloma. The mean area of graft size was 156 ± 120 mm2. Postoperatively, the graft was well tolerated with no failure, discomfort, or dehiscence. During the 17-month mean follow-up, symblepharon, which was clinically nonsignificant, developed in 3 (11%) patients and partial stem cell deficiency was noted in 5 (18%) patients. Cryopreserved ultra-thick human AM is a well-tolerated, effective graft material that is easy to handle. It is a viable alternative for conjunctival surface reconstruction after excision of a conjunctival tumor.

Design of a colicin E7 based chimeric zinc-finger nuclease

Science.gov (United States)

Németh, Eszter; Schilli, Gabriella K.; Nagy, Gábor; Hasenhindl, Christoph; Gyurcsik, Béla; Oostenbrink, Chris

2014-08-01

Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity—offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region.

Antitumor activity od apoptotic nuclease TBN1 from L. esculentum

Czech Academy of Sciences Publication Activity Database

Matoušek, Jaroslav; Podzimek, Tomáš; Poučková, P.; Stehlík, Jan; Škvor, J.; Lipovová, P.; Matoušek, Josef

2010-01-01

Roč. 57, č. 4 (2010), s. 339-348 ISSN 0028-2685 R&D Projects: GA ČR GA521/06/1149; GA ČR GA521/09/1214 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z50450515 Keywords : anticancerogenic and antiproliferative nuclease * dsRNase * human solid malignant tumors Subject RIV: FD - Oncology ; Hematology Impact factor: 1.449, year: 2010

Generation of knockout rabbits using transcription activator-like effector nucleases

OpenAIRE

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

2014-01-01

Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large ...

In vivo excision of pyrimidine dimers is mediated by a DNA N-glycosylase in Micrococcus luteus but not in human fibroblasts

International Nuclear Information System (INIS)

La Belle, M.; Linn, S.

1982-01-01

It has been previously shown that Micrococcus luteus possesses a pyrimidine dimer-specific endonuclease which in vitro, functions as both an endonuclease and DNA-glycosylase. To determine if these combined activities function in vivo, the excision products of UV-irradiated M. luteus were isolated and examined. In addition, a procedure was devised to isolate and examine the excision products from UV-irradiated human fibroblasts to determine if an endonuclease/glycosylase activity functions in the excision of UV-induced pyrimidine dimers in human fibroblasts. It was shown that, in vivo, an endonuclease/glycosylase mechanism is utilized extensively in the repair of pyrimidine dimers by M. luteus, but that human fibroblasts do not appear to use this mechanism. (author)

In vivo excision of pyrimidine dimers is mediated by a DNA N-glycosylase in Micrococcus luteus but not in human fibroblasts

Energy Technology Data Exchange (ETDEWEB)

La Belle, M; Linn, S [California Univ., Berkeley (USA). Dept. of Biochemistry

1982-09-01

It has been previously shown that Micrococcus luteus possesses a pyrimidine dimer-specific endonuclease which in vitro, functions as both an endonuclease and DNA-glycosylase. To determine if these combined activities function in vivo, the excision products of UV-irradiated M. luteus were isolated and examined. In addition, a procedure was devised to isolate and examine the excision products from UV-irradiated human fibroblasts to determine if an endonuclease/glycosylase activity functions in the excision of UV-induced pyrimidine dimers in human fibroblasts. It was shown that, in vivo, an endonuclease/glycosylase mechanism is utilized extensively in the repair of pyrimidine dimers by M. luteus, but that human fibroblasts do not appear to use this mechanism.

DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease

Energy Technology Data Exchange (ETDEWEB)

Dupuy, Aurélie [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Sarasin, Alain, E-mail: alain.sarasin@gustaveroussy.fr [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Service de Génétique, Institut Gustave Roussy (France)

2015-06-15

Graphical abstract: - Highlights: • Full correction of mutation in the XPC gene by engineered nucleases. • Meganucleases and TALENs are inhibited by 5-MeC for inducing double strand breaks. • Gene therapy of XP cells is possible using homologous recombination for DSB repair. - Abstract: Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.

DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease

International Nuclear Information System (INIS)

Dupuy, Aurélie; Sarasin, Alain

2015-01-01

Graphical abstract: - Highlights: • Full correction of mutation in the XPC gene by engineered nucleases. • Meganucleases and TALENs are inhibited by 5-MeC for inducing double strand breaks. • Gene therapy of XP cells is possible using homologous recombination for DSB repair. - Abstract: Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients

DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease.

Science.gov (United States)

Dupuy, Aurélie; Sarasin, Alain

2015-06-01

Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients. Copyright © 2014 Elsevier B.V. All rights reserved.

S1 nuclease from Aspergillus oryzae for the detection of DNA damage and repair in the gamma-irradiated intracerebral rat gliosarcoma 9L

International Nuclear Information System (INIS)

Gutin, P.H.; Hilton, J.; Fein, V.J.; Allen, A.E.; Walker, M.D.

1977-01-01

DNA damage and repair in a rat brain tumor following irradiation in vivo were measured by analysis of the rate of strand separation of the tumor DNA in alkali. Tumors were removed after irradiation and mechanically dissociated to a cellular suspension. Tumor cells were injected into alkali (pH 12) for 20 min at 22 0 C. The fraction of tumor DNA remaining double-stranded after this exposure to alkali was determined by its resistance to S 1 nuclease from Aspergillus oryzae. Double-stranded DNA remains (after enzyme exposure) acid-precipitable for fluorescent assay. The double-stranded fraction after exposure to alkali decreases with increasing radiation dose following first-order kinetics. DNA from tumors excised at intervals after irradiation showed a greater double-stranded fraction in alkali than that from tumors excised immediately, indicating repair of single-strand breaks. Repair of damage produced by 600 rad proceeded with a half-time of approximately 15 min

Evidence for an involvement of thymidine kinase in the excision repair of ultraviolet-irradiated herpes simplex virus in human cells

International Nuclear Information System (INIS)

Intine, R.V.; Rainbow, A.J.

1990-01-01

A wild-type strain of herpes simplex virus type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) and a tk- mutant strain (HSV-1:PTK3B) were used to study the role of the viral tk in the repair of UV-irradiated HSV-1 in human cells. UV survival of HSV-1:PTK3B was substantially reduced compared with that of HSV-1:KOS when infecting normal human cells. In contrast, the UV survival of HSV-1:PTK3B was similar to that of HSV-1:KOS when infecting excision repair-deficient cells from a xeroderma pigmentosum patient from complementation group A. These results suggest that the repair of UV-irradiated HSV-1 in human cells depends, in part at least, on expression of the viral tk and that the repair process influenced by tk activity is excision repair or a process dependent on excision repair

Chemical Approach to Biological Safety: Molecular-Level Control of an Integrated Zinc Finger Nuclease

DEFF Research Database (Denmark)

Németh, Eszter; Asaka, Masamitsu N; Kato, Kohsuke

2018-01-01

circular dichroism spectroscopy, and nano-electrospray ionisation mass spectrometry. In situ intramolecular activation of the nuclease domain was observed, resulting in specific cleavage of DNA with moderate activity. This study represents a new approach to AN design through integrated nucleases consisting......Application of artificial nucleases (ANs) in genome editing is still hindered by their cytotoxicity related to off-target cleavages. This problem can be targeted by regulation of the nuclease domain. Here, we provide an experimental survey of computationally designed integrated zinc finger...... nucleases, constructed by linking the inactivated catalytic centre and the allosteric activator sequence of the colicin E7 nuclease domain to the two opposite termini of a zinc finger array. DNA specificity and metal binding were confirmed by electrophoretic mobility shift assays, synchrotron radiation...

Proviral HIV-genome-wide and pol-gene specific Zinc Finger Nucleases: Usability for targeted HIV gene therapy

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Wayengera Misaki

2011-07-01

Full Text Available Abstract Background Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that ex-vivo or even in-vivo abolition or disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively. However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i to assemble and construct zinc finger arrays and nucleases (ZFN with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii to advance a model for pre-clinically testing lentiviral vectors (LV that deliver and transduce either ZFN genotype. Methods and Results First, we computationally generated the consensus sequences of (a 114 dsDNA-binding zinc finger (Zif arrays (ZFAs or ZifHIV-pol and (b two zinc-finger nucleases (ZFNs which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (ZifHIV-polFN. Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (ZifHIV-1FN. Second, a model for constructing lentiviral vectors (LVs that deliver and transduce a diploid copy of either ZifHIV-polFN or ZifHIV-1FN chimeric genes (termed LV- 2xZifHIV-polFN and LV- 2xZifHIV-1FN, respectively is proposed. Third, two preclinical models for controlled testing of

Proviral HIV-genome-wide and pol-gene specific zinc finger nucleases: usability for targeted HIV gene therapy.

Science.gov (United States)

Wayengera, Misaki

2011-07-22

Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that ex-vivo or even in-vivo abolition or disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases) AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively). However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX) at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i) to assemble and construct zinc finger arrays and nucleases (ZFN) with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii) to advance a model for pre-clinically testing lentiviral vectors (LV) that deliver and transduce either ZFN genotype. First, we computationally generated the consensus sequences of (a) 114 dsDNA-binding zinc finger (Zif) arrays (ZFAs or ZifHIV-pol) and (b) two zinc-finger nucleases (ZFNs) which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (ZifHIV-polFN). Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (ZifHIV-1FN). Second, a model for constructing lentiviral vectors (LVs) that deliver and transduce a diploid copy of either ZifHIV-polFN or ZifHIV-1FN chimeric genes (termed LV- 2xZifHIV-polFN and LV- 2xZifHIV-1FN, respectively) is proposed. Third, two preclinical models for controlled testing of the safety and efficacy of either of these

Prediction of Human Pharmacokinetic Profile After Transdermal Drug Application Using Excised Human Skin.

Science.gov (United States)

Yamamoto, Syunsuke; Karashima, Masatoshi; Arai, Yuta; Tohyama, Kimio; Amano, Nobuyuki

2017-09-01

Although several mathematical models have been reported for the estimation of human plasma concentration profiles of drug substances after dermal application, the successful cases that can predict human pharmacokinetic profiles are limited. Therefore, the aim of this study is to investigate the prediction of human plasma concentrations after dermal application using in vitro permeation parameters obtained from excised human skin. The in vitro skin permeability of 7 marketed drug products was evaluated. The plasma concentration-time profiles of the drug substances in humans after their dermal application were simulated using compartment models and the clinical pharmacokinetic parameters. The transdermal process was simulated using the in vitro skin permeation rate and lag time assuming a zero-order absorption. These simulated plasma concentration profiles were compared with the clinical data. The result revealed that the steady-state plasma concentration of diclofenac and the maximum concentrations of nicotine, bisoprolol, rivastigmine, and lidocaine after topical application were within 2-fold of the clinical data. Furthermore, the simulated concentration profiles of bisoprolol, nicotine, and rivastigmine reproduced the decrease in absorption due to drug depletion from the formulation. In conclusion, this simple compartment model using in vitro human skin permeation parameters as zero-order absorption predicted the human plasma concentrations accurately. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

A sensitive assay for Staphylococcus aureus nucleases

Energy Technology Data Exchange (ETDEWEB)

Kohli, J K; Vakil, B V; Patil, M S; Pandey, V N; Pradhan, D S [Bhabha Atomic Reserach Centre, Bombay (India). Biochemistry Div.

1989-10-01

A sensitive assay for staphylococcal nuclease involving incubation of the enzyme sample with heat-denatured ({sup 3}H) thymidine labelled DNA from E.coli, precipitation with trichloroacetic acid and measurement of the radioactivity of acid-soluble nucleotides released has been developed. The assay is sensitive enough to be used for comparing the levels of nucleases elaborated by different strains of S. aureus as well as for determining the extent of contamination of S. aureus in food and water samples even at levels at which the conventional spectrophotometric and toluidine blue-DNA methods are totally inadequate. (author). 26 refs., 3 figs ., 3 tabs.

Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis.

Science.gov (United States)

Almeida Garcia, Rayssa; Lima Pepino Macedo, Leonardo; Cabral do Nascimento, Danila; Gillet, François-Xavier; Moreira-Pinto, Clidia Eduarda; Faheem, Muhammad; Moreschi Basso, Angelina Maria; Mattar Silva, Maria Cristina; Grossi-de-Sa, Maria Fatima

2017-01-01

RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (Anthonomus grandis) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted AgraNuc1, AgraNuc2, and AgraNuc3, and the influences of these nucleases on the gene silencing of A. grandis chitin synthase II (AgraChSII) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the AgraChSII gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that A. grandis midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests.

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

Science.gov (United States)

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

2014-04-24

Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in 'all-in-one' lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases.DOI: http://dx.doi.org/10.7554/eLife.01911.001. Copyright © 2014, Cai et al.

Aggregation of fragmented chromatin associated with the appearance of products of its nuclease treatment

International Nuclear Information System (INIS)

Lobanenkov, V.V.; Mironov, N.M.; Kupriyanova, E.I.; Shapot, V.S.

1986-01-01

Isolated cell nuclei were incubated with nucleases, and then the chromatin was extracted with a low-salt buffer. When degradation of the nuclear chromatin DNase I or micrococcal nuclease is intensified, solubilization of the deoxyribonucleoprotein (DNP) in low-salt buffer at first increases, reaching a maximum in the case of hydrolysis of 2-4% of the nuclear DNA, but after intensive treatment with nucleases, it decreases sharply. Soluble fragmented chromatin is aggregated during treatment with DNase I. The addition of exogenous products of nuclease treatment of isolated nuclei to a preparation of gelatinous chromatin induces its aggregation. Pretreatment of nuclear chromatin with RNase prevents the solubilization of DNP by solutions with low ionic strength. Certain experimental data obtained using rigorous nuclease treatment are discussed; for their interpretation it is necessary to consider the effect of aggregation of fragmented chromatin by products of its nuclease degradation

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.

Science.gov (United States)

Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

2014-08-01

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner. © 2014 Remy et al.; Published by Cold Spring Harbor Laboratory Press.

Transcriptional and post-transcriptional regulation of nucleotide excision repair genes in human cells

Energy Technology Data Exchange (ETDEWEB)

Lefkofsky, Hailey B. [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Veloso, Artur [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI (United States); Bioinformatics Program, Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI (United States); Ljungman, Mats, E-mail: ljungman@umich.edu [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI (United States)

2015-06-15

Nucleotide excision repair (NER) removes DNA helix-distorting lesions induced by UV light and various chemotherapeutic agents such as cisplatin. These lesions efficiently block the elongation of transcription and need to be rapidly removed by transcription-coupled NER (TC-NER) to avoid the induction of apoptosis. Twenty-nine genes have been classified to code for proteins participating in nucleotide excision repair (NER) in human cells. Here we explored the transcriptional and post-transcriptional regulation of these NER genes across 13 human cell lines using Bru-seq and BruChase-seq, respectively. Many NER genes are relatively large in size and therefore will be easily inactivated by UV-induced transcription-blocking lesions. Furthermore, many of these genes produce transcripts that are rather unstable. Thus, these genes are expected to rapidly lose expression leading to a diminished function of NER. One such gene is ERCC6 that codes for the CSB protein critical for TC-NER. Due to its large gene size and high RNA turnover rate, the ERCC6 gene may act as dosimeter of DNA damage so that at high levels of damage, ERCC6 RNA levels would be diminished leading to the loss of CSB expression, inhibition of TC-NER and the promotion of cell death.

Efficiency of repair of pyrimidine dimers and psoralen monoadducts in normal and xeroderma pigmentosum human cells

International Nuclear Information System (INIS)

Cleaver, J.E.; Charles, W.C.; Kong, S.H.

1984-01-01

Repair of DNA damage produced by ultraviolet light or 5-methylisopsoralen in normal and xeroderma pigmentosum human cells involves many similar steps. Aphidicolin and cytosine arabinoside block repair of both kinds of damage with similar efficiency, indicating that DNA polymerase α has a major role in repair for these lesions. In xeroderma pigmentosum cells of various complementation groups, the relative efficiency of excision repair for both ultraviolet- and 5-methylisopsoralen-induced damage was group A< C< D, indicating a close resemblance between both kinds of lesions in relation to the repair deficiencies in these groups. At high doses, the maximum rate of repair of damage by ultraviolet light was about twice that for methylisopsoralen damage, possibly because ultraviolet-induced damage forms a substrate that is more readily recognized and excised than that of the psoralen adducts. Differences in the structural distortions to DNA caused by these kinds of damage could be detected using single strand specific nucleases which excised dimers but not 5-MIP adducts from double strand DNA. (author)

Hybrid nanosensor for colorimetric and ultrasensitive detection of nuclease contaminations

Science.gov (United States)

Cecere, Paola; Valentini, Paola; Pompa, Pier Paolo

2016-04-01

Nucleases are ubiquitous enzymes that degrade DNA or RNA, thus they can prejudice the good outcome of molecular biology experiments involving nucleic acids. We propose a colorimetric test for the naked-eye detection of nuclease contaminations. The system uses an hybrid nanosensor, based on gold nanoparticles functionalized with DNA probes. Our assay is rapid, instrument-free, simple and low-cost. Moreover, it reaches sensitivity equal or better than those of commercial kits, and presents a lot of advantageous aspects. Therefore, it is very competitive, with a real market potential. This test will be relevant in routine process monitoring in scientific laboratories, and in quality control in clinical laboratories and industrial processes, allowing the simultaneous detection of nucleases with different substrate specificities and large-scale screening.

Plant plasma membrane-bound staphylococcal-like DNases as a novel class of eukaryotic nucleases

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Leśniewicz Krzysztof

2012-10-01

Full Text Available Abstract Background The activity of degradative nucleases responsible for genomic DNA digestion has been observed in all kingdoms of life. It is believed that the main function of DNA degradation occurring during plant programmed cell death is redistribution of nucleic acid derived products such as nitrogen, phosphorus and nucleotide bases. Plant degradative nucleases that have been studied so far belong mainly to the S1-type family and were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application. However, the explanation of how degraded DNA components are exported from the dying cells for further reutilization remains open. Results Bioinformatic and experimental data presented in this paper indicate that two Arabidopsis staphylococcal-like nucleases, named CAN1 and CAN2, are anchored to the cell membrane via N-terminal myristoylation and palmitoylation modifications. Both proteins possess a unique hybrid structure in their catalytic domain consisting of staphylococcal nuclease-like and tRNA synthetase anticodon binding-like motifs. They are neutral, Ca2+-dependent nucleaces showing a different specificity toward the ssDNA, dsDNA and RNA substrates. A study of microarray experiments and endogenous nuclease activity revealed that expression of CAN1 gene correlates with different forms of programmed cell death, while the CAN2 gene is constitutively expressed. Conclusions In this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. The identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for DNA and RNA degradation. The potential functions of these nucleases in relation to their unique intracellular location are discussed.

Halophilic Nuclease from a Moderately Halophilic Micrococcus varians

Science.gov (United States)

Kamekura, Masahiro; Onishi, Hiroshi

1974-01-01

The moderately halophilic bacterium Micrococcus varians, isolated from soy sauce mash, produced extracellular nuclease when cultivated aerobically in media containing 1 to 4 M NaCl or KCl. The enzyme, purified to an electrophoretically homogeneous state, had both ribonuclease and deoxyribonuclease activities. The nuclease had maximal activity in the presence of 2.9 M NaCl or 2.1 M KCl at 40 C. The enzymatic activity was lost by dialysis against low-salt buffer, whereas when the inactivated enzyme was dialyzed against 3.4 M NaCl buffer as much as 77% of the initial activity could be restored. Images PMID:4852218

Analysis of pyrimidine dimer content of isolated DNA by nuclease digestion

International Nuclear Information System (INIS)

Farland, W.H.; Sutherland, B.M.

1980-01-01

Isolated DNA is highly susceptible to degradation by exogenous nucleases. Complete digestion is possible with a number of well-characterized enzymes from a variety of sources. Treatment of DNA with a battery of enzymes including both phosphodiesterase and phosphatase activities yields a mixture of nucleosides and inorganic phosphate (P/sub i/) as a final product. Unlike native DNA, ultraviolet-irradiated DNA is resistant to complete digestion. Setlow et al. demonstrated that the structural changes in the DNA responsible for the nuclease resistance were the formation of cyclobutyl pyrimidine dimers, the major photoproduct in UV-irradiated DNA. Using venom phosphodiesterase, they demonstrated that UV irradiation of DNA affected both the rate and extent of enzymatic hydrolysis. In addition, it was demonstrated that the major nuclease-resistant product of this hydrolysis was an oligonucleotide containing dimerized pyrimidines. Treatment of the DNA to split the dimers, either photochemically or photoenzymatically, rendered the polymer more susceptible to hydrolysis by the phosphodiesterase. The specificity of photoreactivating enzyme for pyrimidine dimers lends support to the role of these structures in conferring nuclease resistance to UV-irradiated DNA. The nuclease resistance of DNA containing dimers has been the basis of several assays for the measurement of these photoproducts. Sutherland and Chamberlin reported the development of a rapid and sensitive assay for dimers in 32 P-labeled DNA

Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps.

Science.gov (United States)

Doke, M; Fukamachi, H; Morisaki, H; Arimoto, T; Kataoka, H; Kuwata, H

2017-08-01

Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net-like fibers comprising DNA and antimicrobial components such as histones, LL-37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram-negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA- and nucD-encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg 2+ and Ca 2+ for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs. © 2016 The Authors Molecular Oral Microbiology Published by John Wiley & Sons Ltd.

Cell wall-anchored nuclease of Streptococcus sanguinis contributes to escape from neutrophil extracellular trap-mediated bacteriocidal activity.

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Chisato Morita

Full Text Available Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease, and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs. Recombinant SWAN protein (rSWAN digested multiple forms of DNA including NET DNA and human RNA, which required both Mg(2+ and Ca(2+ for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression.

Cell wall-anchored nuclease of Streptococcus sanguinis contributes to escape from neutrophil extracellular trap-mediated bacteriocidal activity.

Science.gov (United States)

Morita, Chisato; Sumioka, Ryuichi; Nakata, Masanobu; Okahashi, Nobuo; Wada, Satoshi; Yamashiro, Takashi; Hayashi, Mikako; Hamada, Shigeyuki; Sumitomo, Tomoko; Kawabata, Shigetada

2014-01-01

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg(2+) and Ca(2+) for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression.

Crispr-mediated Gene Targeting of Human Induced Pluripotent Stem Cells.

Science.gov (United States)

Byrne, Susan M; Church, George M

2015-01-01

CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem or induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, we describe an optimized protocol for genome engineering of human iPSCs using a simple transient transfection of plasmids and/or single-stranded oligonucleotides. With this protocol, we achieve transfection efficiencies greater than 60%, with gene disruption efficiencies from 1-25% and gene insertion/replacement efficiencies from 0.5-10% without any further selection or enrichment steps. We also describe how to design and assess optimal sgRNA target sites and donor targeting vectors; cloning individual iPSC by single cell FACS sorting, and genotyping successfully edited cells.

A comprehensive overview of computational resources to aid in precision genome editing with engineered nucleases.

Science.gov (United States)

Periwal, Vinita

2017-07-01

Genome editing with engineered nucleases (zinc finger nucleases, TAL effector nucleases s and Clustered regularly inter-spaced short palindromic repeats/CRISPR-associated) has recently been shown to have great promise in a variety of therapeutic and biotechnological applications. However, their exploitation in genetic analysis and clinical settings largely depends on their specificity for the intended genomic target. Large and complex genomes often contain highly homologous/repetitive sequences, which limits the specificity of genome editing tools and could result in off-target activity. Over the past few years, various computational approaches have been developed to assist the design process and predict/reduce the off-target activity of these nucleases. These tools could be efficiently used to guide the design of constructs for engineered nucleases and evaluate results after genome editing. This review provides a comprehensive overview of various databases, tools, web servers and resources for genome editing and compares their features and functionalities. Additionally, it also describes tools that have been developed to analyse post-genome editing results. The article also discusses important design parameters that could be considered while designing these nucleases. This review is intended to be a quick reference guide for experimentalists as well as computational biologists working in the field of genome editing with engineered nucleases. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Genome Editing in Human Pluripotent Stem Cells.

Science.gov (United States)

Carlson-Stevermer, Jared; Saha, Krishanu

2017-01-01

Genome editing in human pluripotent stem cells (hPSCs) enables the generation of reporter lines and knockout cell lines. Zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 technology have recently increased the efficiency of proper gene editing by creating double strand breaks (DSB) at defined sequences in the human genome. These systems typically use plasmids to transiently transcribe nucleases within the cell. Here, we describe the process for preparing hPSCs for transient expression of nucleases via electroporation and subsequent analysis to create genetically modified stem cell lines.

GUIDEseq: a bioconductor package to analyze GUIDE-Seq datasets for CRISPR-Cas nucleases.

Science.gov (United States)

Zhu, Lihua Julie; Lawrence, Michael; Gupta, Ankit; Pagès, Hervé; Kucukural, Alper; Garber, Manuel; Wolfe, Scot A

2017-05-15

Genome editing technologies developed around the CRISPR-Cas9 nuclease system have facilitated the investigation of a broad range of biological questions. These nucleases also hold tremendous promise for treating a variety of genetic disorders. In the context of their therapeutic application, it is important to identify the spectrum of genomic sequences that are cleaved by a candidate nuclease when programmed with a particular guide RNA, as well as the cleavage efficiency of these sites. Powerful new experimental approaches, such as GUIDE-seq, facilitate the sensitive, unbiased genome-wide detection of nuclease cleavage sites within the genome. Flexible bioinformatics analysis tools for processing GUIDE-seq data are needed. Here, we describe an open source, open development software suite, GUIDEseq, for GUIDE-seq data analysis and annotation as a Bioconductor package in R. The GUIDEseq package provides a flexible platform with more than 60 adjustable parameters for the analysis of datasets associated with custom nuclease applications. These parameters allow data analysis to be tailored to different nuclease platforms with different length and complexity in their guide and PAM recognition sequences or their DNA cleavage position. They also enable users to customize sequence aggregation criteria, and vary peak calling thresholds that can influence the number of potential off-target sites recovered. GUIDEseq also annotates potential off-target sites that overlap with genes based on genome annotation information, as these may be the most important off-target sites for further characterization. In addition, GUIDEseq enables the comparison and visualization of off-target site overlap between different datasets for a rapid comparison of different nuclease configurations or experimental conditions. For each identified off-target, the GUIDEseq package outputs mapped GUIDE-Seq read count as well as cleavage score from a user specified off-target cleavage score prediction

Isolation of the functional human excision repair gene ERCC5 by intercosmid recombination

International Nuclear Information System (INIS)

Mudgett, J.S.; MacInnes, M.A.

1990-01-01

The complete human nucleotide exicision repair gene ERCC5 was isolated as a functional gene on overlapping cosmids. ERCC5 corrects the excision repair deficiency of Chinese hamster ovary cell line UV135, of complementation group 5. Cosmids that contained human sequences were obtained from a UV-resistant cell line derived from UV135 cells transformed with human genomic DNA. Individually, none of the cosmids complemented the UV135 repair defect; cosmid groups were formed to represent putative human genomic regions, and specific pairs of cosmids that effectively transformed UV135 cells to UV resistance were identified. Analysis of transformants derived from the active cosmid pairs showed that the functional 32-kbp ERCC5 gene was reconstructed by homologous intercosmid recombination. The cloned human sequences exhibited 100% concordance with the locus designated genetically as ERCC5 located on human chromosome 13q. Cosmid-transformed UV135 host cells repaired cytotoxic damage to levels about 70% of normal and repaired UV-irradiated shuttle vector DNA to levels about 82% of normal

Efficient Genome Editing in Induced Pluripotent Stem Cells with Engineered Nucleases In Vitro.

Science.gov (United States)

Termglinchan, Vittavat; Seeger, Timon; Chen, Caressa; Wu, Joseph C; Karakikes, Ioannis

2017-01-01

Precision genome engineering is rapidly advancing the application of the induced pluripotent stem cells (iPSCs) technology for in vitro disease modeling of cardiovascular diseases. Targeted genome editing using engineered nucleases is a powerful tool that allows for reverse genetics, genome engineering, and targeted transgene integration experiments to be performed in a precise and predictable manner. However, nuclease-mediated homologous recombination is an inefficient process. Herein, we describe the development of an optimized method combining site-specific nucleases and the piggyBac transposon system for "seamless" genome editing in pluripotent stem cells with high efficiency and fidelity in vitro.

Nucleotide excision repair in yeast

NARCIS (Netherlands)

Eijk, Patrick van

2012-01-01

Nucleotide Excision Repair (NER) is a conserved DNA repair pathway capable of removing a broad spectrum of DNA damage. In human cells a defect in NER leads to the disorder Xeroderma pigmentosum (XP). The yeast Saccharomyces cerevisiae is an excellent model organism to study the mechanism of NER. The

Mislocalization of XPF-ERCC1 nuclease contributes to reduced DNA repair in XP-F patients.

Directory of Open Access Journals (Sweden)

Anwaar Ahmad

2010-03-01

Full Text Available Xeroderma pigmentosum (XP is caused by defects in the nucleotide excision repair (NER pathway. NER removes helix-distorting DNA lesions, such as UV-induced photodimers, from the genome. Patients suffering from XP exhibit exquisite sun sensitivity, high incidence of skin cancer, and in some cases neurodegeneration. The severity of XP varies tremendously depending upon which NER gene is mutated and how severely the mutation affects DNA repair capacity. XPF-ERCC1 is a structure-specific endonuclease essential for incising the damaged strand of DNA in NER. Missense mutations in XPF can result not only in XP, but also XPF-ERCC1 (XFE progeroid syndrome, a disease of accelerated aging. In an attempt to determine how mutations in XPF can lead to such diverse symptoms, the effects of a progeria-causing mutation (XPF(R153P were compared to an XP-causing mutation (XPF(R799W in vitro and in vivo. Recombinant XPF harboring either mutation was purified in a complex with ERCC1 and tested for its ability to incise a stem-loop structure in vitro. Both mutant complexes nicked the substrate indicating that neither mutation obviates catalytic activity of the nuclease. Surprisingly, differential immunostaining and fractionation of cells from an XFE progeroid patient revealed that XPF-ERCC1 is abundant in the cytoplasm. This was confirmed by fluorescent detection of XPF(R153P-YFP expressed in Xpf mutant cells. In addition, microinjection of XPF(R153P-ERCC1 into the nucleus of XPF-deficient human cells restored nucleotide excision repair of UV-induced DNA damage. Intriguingly, in all XPF mutant cell lines examined, XPF-ERCC1 was detected in the cytoplasm of a fraction of cells. This demonstrates that at least part of the DNA repair defect and symptoms associated with mutations in XPF are due to mislocalization of XPF-ERCC1 into the cytoplasm of cells, likely due to protein misfolding. Analysis of these patient cells therefore reveals a novel mechanism to potentially

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

DEFF Research Database (Denmark)

Juillerat, Alexandre; Dubois, Gwendoline; Valton, Julien

2014-01-01

A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within...... their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator......-like effector nucleases (TALEN). The analysis of >15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only...

Potential relationships between morphological differentiation and mutants with high nuclease P1 production of Penicillium citrinum

Energy Technology Data Exchange (ETDEWEB)

Xinle, Liang; Qian, Shou; Hong, Zhang; Min, Chen [Department of Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang (China); Xuan, Liu [Beihai Institute of Environmental Science, Beihai, Guangxi (China)

2009-08-15

Diversification of colony characteristics of mutants derived from Penicillium citrinum CICC 4011 treated with {sup 60}Co {gamma}-irradiation and protoplast fusion were analyzed. There were distinct differences among mutants with different nuclease P1 activity, especially in pigment productivity. Color of colony was changed from the original green to white, grey-green, or yellow-green etc., while the nuclease P1 activity would be fluctuated with the color change. The hypothesis was suggested that there would be a relationship between pigments and nuclease P1 production. Mutants with grey-green colony would give out high nuclease P1 outputs in a high probability such as mutant J1Y6 (nuclease P1 activity, 167.3U/ml) and fusant F-13 (nuclease P1 activity, 568.7U/ml), while others with deep-green colony observed low nuclease outputs. Four variation strains didn't show any significant difference in growth rate. Broom branches of conidiophore stem in J1Y6 and F-13 were obviously reduced, conidiophores productivity reduced, but hyphae growth haled. These suggested that nuclease P1 production was associated with growth phase, but pigment synthesis course wasn't. RAPD from 6 randomly selected primers was used to analyze the polymorphic rich of the four strains, the results showed that there were 70 percent polymorphism detection rate among those. UPGMA cluster analysis and genetic map constructed by NTSYS-PC software, which showed that J1Y6 and F-14 were clustered as one group at similar coefficient 0.9, where there was an appear distance from the group of 4011 and F-R-33 strains (similar coefficient 0.8). (authors)

Potential relationships between morphological differentiation and mutants with high nuclease P1 production of Penicillium citrinum

International Nuclear Information System (INIS)

Liang Xinle; Shou Qian; Zhang Hong; Chen Min; Liu Xuan

2009-01-01

Diversification of colony characteristics of mutants derived from Penicillium citrinum CICC 4011 treated with 60 Co γ-irradiation and protoplast fusion were analyzed. There were distinct differences among mutants with different nuclease P1 activity, especially in pigment productivity. Color of colony was changed from the original green to white, grey-green, or yellow-green etc., while the nuclease P1 activity would be fluctuated with the color change. The hypothesis was suggested that there would be a relationship between pigments and nuclease P1 production. Mutants with grey-green colony would give out high nuclease P1 outputs in a high probability such as mutant J1Y6( nuclease P1 activity, 167.3U/ml) and fusant F-13 (nuclease P1 activity, 568.7U/ml), while others with deep-green colony observed low nuclease outputs. Four variation strains didn't show any significant difference in growth rate. Broom branches of conidiophore stem in J1Y6 and F-13 were obviously reduced, conidiophores productivity reduced, but hyphae growth haled. These suggested that nuclease P1 production was associated with growth phase, but pigment synthesis course wasn't. RAPD from 6 randomly selected primers was used to analyze the polymorphic rich of the four strains, the results showed that there were 70 percent polymorphism detection rate among those. UPGMA cluster analysis and genetic map constructed by NTSYS-PC software, which showed that J1Y6 and F-14 were clustered as one group at similar coefficient 0.9, where there was an appear distance from the group of 4011 and F-R-33 strains (similar coefficient 0.8). (authors)

Improving Fab' fragment retention in an autonucleolytic Escherichia coli strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA.

Science.gov (United States)

Schofield, Desmond M; Sirka, Ernestas; Keshavarz-Moore, Eli; Ward, John M; Nesbeth, Darren N

2017-12-01

To reduce unwanted Fab' leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab' fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives. We attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab' grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab' coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab' leakage relative to the original autonucleolytic Fab' strain with OmpA-fused staphylococcal nuclease. We successfully rescued Fab' leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab' fragment to the surrounding growth media.

Removal of misincorporated ribonucleotides from prokaryotic genomes: an unexpected role for nucleotide excision repair.

Directory of Open Access Journals (Sweden)

Alexandra Vaisman

2013-11-01

Full Text Available Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER. We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8(th phosphodiester bond 5' and 4(th-5(th phosphodiester bonds 3' of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be

Dna2 nuclease-helicase structure, mechanism and regulation by Rpa.

Science.gov (United States)

Zhou, Chun; Pourmal, Sergei; Pavletich, Nikola P

2015-11-02

The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5' end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5' but not 3' end, explaining how Rpa regulates cleavage polarity.

Surgical excision for recurrent herpes simplex virus 2 (HSV-2) anogenital infection in a patient with human immunodeficiency virus (HIV).

Science.gov (United States)

Arinze, Folasade; Shaver, Aaron; Raffanti, Stephen

2017-10-01

Recurrent anogenital herpes simplex virus infections are common in patients with human immunodeficiency virus (HIV), of whom approximately 5% develop resistance to acyclovir. We present a case of a 49-year-old man with HIV who had an 8-year history of recurrent left inguinal herpes simplex virus type 2 ulcerations. He initially responded to oral acyclovir, but developed resistance to acyclovir and eventually foscarnet. The lesion progressed to a large hypertrophic mass that required surgical excision, which led to resolution without recurrences. Our case highlights the importance of surgical excision as a treatment option in refractory herpes simplex virus anogenital infections.

Phylogenomic analysis of the GIY-YIG nuclease superfamily

Directory of Open Access Journals (Sweden)

Bujnicki Janusz M

2006-04-01

Full Text Available Abstract Background The GIY-YIG domain was initially identified in homing endonucleases and later in other selfish mobile genetic elements (including restriction enzymes and non-LTR retrotransposons and in enzymes involved in DNA repair and recombination. However, to date no systematic search for novel members of the GIY-YIG superfamily or comparative analysis of these enzymes has been reported. Results We carried out database searches to identify all members of known GIY-YIG nuclease families. Multiple sequence alignments together with predicted secondary structures of identified families were represented as Hidden Markov Models (HMM and compared by the HHsearch method to the uncharacterized protein families gathered in the COG, KOG, and PFAM databases. This analysis allowed for extending the GIY-YIG superfamily to include members of COG3680 and a number of proteins not classified in COGs and to predict that these proteins may function as nucleases, potentially involved in DNA recombination and/or repair. Finally, all old and new members of the GIY-YIG superfamily were compared and analyzed to infer the phylogenetic tree. Conclusion An evolutionary classification of the GIY-YIG superfamily is presented for the very first time, along with the structural annotation of all (subfamilies. It provides a comprehensive picture of sequence-structure-function relationships in this superfamily of nucleases, which will help to design experiments to study the mechanism of action of known members (especially the uncharacterized ones and will facilitate the prediction of function for the newly discovered ones.

Human lactoferrin efficiently targeted into caprine beta-lactoglobulin locus with transcription activator-like effector nucleases

Directory of Open Access Journals (Sweden)

Yu-Guo Yuan

2017-08-01

Full Text Available Objective To create genetically modified goat as a biopharming source of recombinant human lacotoferrin (hLF with transcription activator-like effector nucleases. Methods TALENs and targeting vector were transferred into cultured fibroblasts to insert hLF cDNA in the goat beta-lactoglobulin (BLG locus with homology-directed repair. The gene targeted efficiency was checked using sequencing and TE7I assay. The bi-allelic gene targeted colonies were isolated and confirmed with polymerase chain reaction, and used as donor cells for somatic cell nuclear transfer (SCNT. Results The targeted efficiency for BLG gene was approximately 10%. Among 12 Bi-allelic gene targeted colonies, five were used in first round SCNT and 4 recipients (23% were confirmed pregnant at 30 d. In second round SCNT, 7 (53%, 4 (31%, and 3 (23% recipients were confirmed to be pregnant by ultrasound on 30 d, 60 d, and 90 d. Conclusion This finding signifies the combined use of TALENs and SCNT can generate bi-allelic knock-in fibroblasts that can be cloned in a fetus. Therefore, it might lay the foundation for transgenic hLF goat generation and possible use of their mammary gland as a bioreactor for large-scale production of recombinant hLF.

Excision repair of bulky lesions in the DNA of mammalian cells

International Nuclear Information System (INIS)

Setlow, R.B.; Grist, E.

1980-01-01

The report examines the process of excision repair of pyrimidine dimers from uv-irradiated and chemically challenged human cells. It is shown by means of a sensitive endonuclease assay that the amount of excision observed depends upon the isotope used to label cells, and that XP heterozygotes are between normals and XPs

Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials.

Science.gov (United States)

Bikard, David; Euler, Chad W; Jiang, Wenyan; Nussenzweig, Philip M; Goldberg, Gregory W; Duportet, Xavier; Fischetti, Vincent A; Marraffini, Luciano A

2014-11-01

Antibiotics target conserved bacterial cellular pathways or growth functions and therefore cannot selectively kill specific members of a complex microbial population. Here, we develop programmable, sequence-specific antimicrobials using the RNA-guided nuclease Cas9 (refs.1,2) delivered by a bacteriophage. We show that Cas9, reprogrammed to target virulence genes, kills virulent, but not avirulent, Staphylococcus aureus. Reprogramming the nuclease to target antibiotic resistance genes destroys staphylococcal plasmids that harbor antibiotic resistance genes and immunizes avirulent staphylococci to prevent the spread of plasmid-borne resistance genes. We also show that CRISPR-Cas9 antimicrobials function in vivo to kill S. aureus in a mouse skin colonization model. This technology creates opportunities to manipulate complex bacterial populations in a sequence-specific manner.

Kinetics of thymine dimer excision in ultraviolet-irradiated human cells

International Nuclear Information System (INIS)

Ehmann, U.K.; Cook, K.H.; Friedberg, E.C.

1978-01-01

We have investigated the kinetics of the loss of thymine dimers from the acid-insoluble fraction of several ultraviolet (uv)-irradiated cultured human cell lines. Our results show that uv fluences between 10 and 40 J/m 2 produce an average of 21 to 85 x 10 5 thymine dimers per cell and an eventual maximal loss per cell of 12 to 20 x 10 5 thymine dimers. The time for half-maximal loss of dimers ranged from 12 to 22 h after uv irradiation. In contrast, the time for half-maximal repair synthesis of DNA measured by autoradiography was 4.5 h. This figure agrees well with reported half-maximal repair synthesis times, which range from 0.5 to 3.6 h based on our analysis. The discrepancy in the kinetics of the loss of thymine dimers from DNA and repair synthesis is discussed in terms of possible molecular mechanisms of thymine dimer excision in vivo and in terms of possible experimental artifacts

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

KAUST Repository

Mahfouz, Magdy M.

2011-01-24

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

Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster

OpenAIRE

Simoni, A; Siniscalchi, C; Chan, Y-S; Huen, DS; Russell, S; Windbichler, N; Crisanti, A

2014-01-01

Selfish genes are DNA elements that increase their rate of genetic transmission at the expense of other genes in the genome and can therefore quickly spread within a population. It has been suggested that selfish elements could be exploited to modify the genome of entire populations for medical and ecological applications. Here we report that transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) can be engineered into site-specific synthetic selfish elements (S...

Modulation of DNA base excision repair during neuronal differentiation

DEFF Research Database (Denmark)

Sykora, Peter; Yang, Jenq-Lin; Ferrarelli, Leslie K

2013-01-01

DNA damage susceptibility and base excision DNA repair (BER) capacity in undifferentiated and differentiated human neural cells. The results show that undifferentiated human SH-SY5Y neuroblastoma cells are less sensitive to oxidative damage than their differentiated counterparts, in part because...

Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing

Science.gov (United States)

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

2016-01-01

The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. PMID:26750397

Eukaryotic zinc-dependent multifunctional nuclease I

Czech Academy of Sciences Publication Activity Database

Koval, Tomáš; Lipovová, P.; Podzimek, T.; Matoušek, Jaroslav; Stránský, Jan; Dušková, Jarmila; Fejfarová, Karla; Skálová, Tereza; Hašek, Jindřich; Dohnálek, Jan

2014-01-01

Roč. 70, Supplement /August/ (2014), C211 ISSN 0108-7673. [Congress and General Assembly of the International Union of Crystallography /23./ - IUCr 2014. 05.08.2014-12.08.2014, Montreal] R&D Projects: GA MŠk(CZ) EE2.3.30.0029; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61389013 ; RVO:86652036 ; RVO:60077344 Keywords : nuclease * tomato * crystal structure Subject RIV: CE - Biochemistry

Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island

International Nuclear Information System (INIS)

Pimkin, Maxim; Miller, C. Glenn; Blakesley, Lauryn; Oleykowski, Catherine A.; Kodali, Nagendra S.; Yeung, Anthony T.

2006-01-01

DNA sequences encoding hypothetical proteins homologous to S1 nuclease from Aspergillus oryzae are found in many organisms including fungi, plants, pathogenic bacteria, and eukaryotic parasites. One of these is the M1 nuclease of Mesorhizobium loti which we demonstrate herein to be an enzymatically active, soluble, and stable S1 homolog that lacks the extensive mannosyl-glycosylation found in eukaryotic S1 nuclease homologs. We have expressed the cloned M1 protein in M. loti and purified recombinant native M1 to near homogeneity and have also isolated a homogeneous M1 carboxy-terminal hexahistidine tag fusion protein. Mass spectrometry and N-terminal Edman degradation sequencing confirmed the protein identity. The enzymatic properties of the purified M1 nuclease are similar to those of S1. At acidic pH M1 is 25 times more active on single-stranded DNA than on double-stranded DNA and 3 times more active on single-stranded DNA than on single-stranded RNA. At neutral pH the RNase activity of M1 exceeds the DNase activity. M1 nicks supercoiled RF-I plasmid DNA and rapidly cuts the phosphodiester bond across from the nick in the resultant relaxed RF-II plasmid DNA. Therefore, M1 represents an active bacterial S1 homolog in spite of great sequence divergence. The biochemical characterization of M1 nuclease supports our sequence alignment that reveals the minimal 21 amino acid residues that are necessarily conserved for the structure and functions of this enzyme family. The ability of M1 to degrade RNA at neutral pH implies previously unappreciated roles of these nucleases in biological systems

Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells.

Science.gov (United States)

Kumar, S; Peng, X; Daley, J; Yang, L; Shen, J; Nguyen, N; Bae, G; Niu, H; Peng, Y; Hsieh, H-J; Wang, L; Rao, C; Stephan, C C; Sung, P; Ira, G; Peng, G

2017-04-17

Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. DNA2 nuclease is an evolutionarily conserved essential enzyme in replication and HR repair. Here we demonstrate that DNA2 is overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90-95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA double strand break end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anticancer drugs by targeting druggable nuclease DNA2. We propose DNA2 inhibition as new strategy in cancer therapy by targeting

Potentiometric sensing of nuclease activities and oxidative damage of single-stranded DNA using a polycation-sensitive membrane electrode.

Science.gov (United States)

Ding, Jiawang; Qin, Wei

2013-09-15

A simple, general and label-free potentiometric method to measure nuclease activities and oxidative DNA damage in a homogeneous solution using a polycation-sensitive membrane electrode is reported. Protamine, a linear polyionic species, is used as an indicator to report the cleavage of DNA by nucleases such as restriction and nonspecific nucleases, and the damage of DNA induced by hydroxyl radicals. Measurements can be done with a titration mode or a direct detection mode. For the potentiometric titration mode, the enzymatic cleavage dramatically affects the electrostatical interaction between DNA and protamine and thus shifts the response curve for the potentiometric titration of the DNA with protamine. Under the optimized conditions, the enzyme activities can be sensed potentiometrically with detection limits of 2.7×10(-4)U/µL for S1 nuclease, and of 3.9×10(-4)U/µL for DNase I. For the direct detection mode, a biocomplex between protamine and DNA is used as a substrate. The nuclease of interest cleaves the DNA from the protamine/DNA complex into smaller fragments, so that free protamine is generated and can be detected potentiometrically via the polycation-sensitive membrane electrode. Using a direct measurement, the nuclease activities could be rapidly detected with detection limits of 3.2×10(-4)U/µL for S1 nuclease, and of 4.5×10(-4)U/µL for DNase I. Moreover, the proposed potentiometric assays demonstrate the potential applications in the detection of hydroxyl radicals. It is anticipated that the present potentiometric strategy will provide a promising platform for high-throughput screening of nucleases, reactive oxygen species and the drugs with potential inhibition abilities. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Directory of Open Access Journals (Sweden)

Finola E Moore

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

Transient correction of excision repair defects in fibroblasts of 9 xeroderma pigmentosum complementation groups by microinjection of crude human cell extract.

NARCIS (Netherlands)

W. Vermeulen (Wim); P. Osseweijer; A.J.R. de Jonge; J.H.J. Hoeijmakers (Jan)

1986-01-01

textabstractCrude extracts from human cells were microinjected into the cytoplasm of cultured fibroblasts from 9 excision-deficient xeroderma pigmentosum (XP) complementation groups. The level of UV-induced unscheduled DNA synthesis (UDS) was measured to determine the effect of the extract on the

Genome editing: a robust technology for human stem cells.

Science.gov (United States)

Chandrasekaran, Arun Pandian; Song, Minjung; Ramakrishna, Suresh

2017-09-01

Human pluripotent stem cells comprise induced pluripotent and embryonic stem cells, which have tremendous potential for biological and therapeutic applications. The development of efficient technologies for the targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. Genome editing of stem cells is possible with the help of synthetic nucleases that facilitate site-specific modification of a gene of interest. Recent advances in genome editing techniques have improved the efficiency and speed of the development of stem cells for human disease models. Zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system are powerful tools for editing DNA at specific loci. Here, we discuss recent technological advances in genome editing with site-specific nucleases in human stem cells.

DNA repair capacity and rate of excision repair in UV-irradiated mammalian cells

International Nuclear Information System (INIS)

Inoue, Masao; Takebe, Hiraku.

1978-01-01

Repair capacities of five mammalian cell strains were measured by colony-forming ability, HCR of UV-irradiated virus, UDS, pyrimidine dimer excision, and semi-conservative DNA replication. Colony-forming ability of UV-irradiated cells was high for human amnion FL cells and mouse L cells, slightly low for African green monkey CV-1 cells, and extremely low for xeroderma pigmentosum cells. HCR of UV-irradiated Herpes simplex virus was high in CV-1 cells, FL and normal human fibroblast cells, low in both XP and L cells. The amount of UDS was high in FL and normal human fibroblast cells, considerably low in CV-1 cells, and essentially no UDS was observed in XP cells. Rate of UDS after UV-irradiation was slower for CV-1 cells than FL and human fibroblast cells. Rate of the excision of thymine-containing dimers from the acid-insoluble fraction during post-irradiation incubation of the cells was rapid in FL and normal human cells and slow in CV-1 cells, and no excision took place in XP cells. Semi-conservative DNA synthesis was reduced after UV-irradiation in all cell lines, but subsequently recovered in FL, normal human and CV-1 cells. The onset of recovery was 4 h after UV-irradiation for FL and normal human cells, but about 6 h for CV-1 cells. The apparent intermediate repair of CV-1 cells except for HCR may be related to the slow rate of excision repair. ''Patch and cut'' model is more favorable than ''cut and patch'' model to elucidate these results. (auth.)

The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

Science.gov (United States)

Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

2017-01-01

Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

Synthesis, X-ray crystal structure, DNA binding and Nuclease activity ...

Indian Academy of Sciences (India)

s12039-016-1125-x. Synthesis, X-ray crystal structure, DNA binding and Nuclease activity of lanthanide(III) complexes of 2-benzoylpyridine acetylhydrazone. KARREDDULA RAJA, AKKILI SUSEELAMMA and KATREDDI HUSSAIN REDDY. ∗.

Arthroscopic excision of ganglion cysts.

Science.gov (United States)

Bontempo, Nicholas A; Weiss, Arnold-Peter C

2014-02-01

Arthroscopy is an advancing field in orthopedics, the applications of which have been expanding over time. Traditionally, excision of ganglion cysts has been done in an open fashion. However, more recently, studies show outcomes following arthroscopic excision to be as good as open excision. Cosmetically, the incisions are smaller and heal faster following arthroscopy. In addition, there is the suggested benefit that patients will regain function and return to work faster following arthroscopic excision. More prospective studies comparing open and arthroscopic excision of ganglion cysts need to be done in order to delineate if there is a true functional benefit. Copyright © 2014 Elsevier Inc. All rights reserved.

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease.

Science.gov (United States)

Doetschman, Thomas; Georgieva, Teodora

2017-03-03

Genetic engineering of model organisms and cultured cells has for decades provided important insights into the mechanisms underlying cardiovascular development and disease. In the past few years the development of several nuclease systems has broadened the range of model/cell systems that can be engineered. Of these, the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has become the favorite for its ease of application. Here we will review this RNA-guided nuclease system for gene editing with respect to its usefulness for cardiovascular studies and with an eye toward potential therapy. Studies on its off-target activity, along with approaches to minimize this activity will be given. The advantages of gene editing versus gene targeting in embryonic stem cells, including the breadth of species and cell types to which it is applicable, will be discussed. We will also cover its use in iPSC for research and possible therapeutic purposes; and we will review its use in muscular dystrophy studies where considerable progress has been made toward dystrophin correction in mice. The CRISPR/Ca9s system is also being used for high-throughput screening of genes, gene regulatory regions, and long noncoding RNAs. In addition, the CRISPR system is being used for nongene-editing purposes such as activation and inhibition of gene expression, as well as for fluorescence tagging of chromosomal regions and individual mRNAs to track their cellular location. Finally, an approach to circumvent the inability of post-mitotic cells to support homologous recombination-based gene editing will be presented. In conclusion, applications of the CRISPR/Cas system are expanding at a breath-taking pace and are revolutionizing approaches to gain a better understanding of human diseases. © 2017 American Heart Association, Inc.

A human beta cell line with drug inducible excision of immortalizing transgenes

Science.gov (United States)

Benazra, Marion; Lecomte, Marie-José; Colace, Claire; Müller, Andreas; Machado, Cécile; Pechberty, Severine; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Solimena, Michele; Scharfmann, Raphaël; Czernichow, Paul; Ravassard, Philippe

2015-01-01

Objectives Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. Methods We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. Results We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. Conclusions EndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture. PMID:26909308

Studies on the molecular mechanism of nucleotide excision repair in human cells

International Nuclear Information System (INIS)

Friedberg, E.C.

1987-01-01

Studies in this laboratory have focused on attempts to define the mechanism of nucleotide excision repair of DNA in human cells, with a view to understanding the molecular pathogenesis of the disease XP. With the advent of recombinant DNA technology, they directed their efforts to the molecular cloning of human genes defective in XP, with a view to using the cloned genes to overexpress proteins of interest for biochemical investigations. Initial studies exploited the selectable phenotype of marked sensitivity to killing of XP group A cells by UV radiation and by other DNA damaging agents. However, except for a single report in 1982 there has been no reproducible demonstration of complementation of the UV sensitivity of XP cells by DNA-mediated transfection. The apparent difficulties associated with transfection of XP cells have been the subject of several recent studies. In view of the multiple problems associated with stable transfection of XP cells using total genomic DNA, they have embarked on an alternative strategy designed to facilitate the cloning of human XP genes. This strategy involves the transfer of single human chromosomes into XP cells and screening for this relatively high frequency event. The idea is to identify chromosomes on which particular XP genes reside and then to isolate non-complementing derivatives of these chromosomes so that highly enriched DNA pools containing genes of interest can be generated by employing one or more subtractive strategies

From hacking the human genome to editing organs.

Science.gov (United States)

Tobita, Takamasa; Guzman-Lepe, Jorge; Collin de l'Hortet, Alexandra

2015-01-01

In the recent decades, human genome engineering has been one of the major interesting research subjects, essentially because it raises new possibilities for personalized medicine and biotechnologies. With the development of engineered nucleases such as the Zinc Finger Nucleases (ZFNs), the Transcription activator-like effector nucleases (TALENs) and more recently the Clustered Regularly Interspaced short Palindromic Repeats (CRISPR), the field of human genome edition has evolved very rapidly. Every new genetic tool is broadening the scope of applications on human tissues, even before we can completely master each of these tools. In this review, we will present the recent advances regarding human genome edition tools, we will discuss the numerous implications they have in research and medicine, and we will mention the limits and concerns about such technologies.

Application of a 5 ' nuclease assay for detection of Lawsonia intracellularis in fecal samples from pigs

DEFF Research Database (Denmark)

Lindecrona, R. H.; Jensen, Tim Kåre; Andersen, P. H.

2002-01-01

A 5' nuclease assay was developed to detect Lawsonia intracellularis in porcine fecal samples. The specific probe and primers were chosen by using the 16S ribosomal DNA gene as a target. The 5' nuclease assay was used with a total of 204 clinical samples, and the results were compared to those of...

Uracil excision repair in Mycobacterium tuberculosis cell-free extracts.

Science.gov (United States)

Kumar, Pradeep; Bharti, Sanjay Kumar; Varshney, Umesh

2011-05-01

Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria. Copyright © 2011 Elsevier Ltd. All rights reserved.

Rapid and Sensitive Detection of Breast Cancer Cells in Patient Blood with Nuclease-Activated Probe Technology

Directory of Open Access Journals (Sweden)

Sven Kruspe

2017-09-01

Full Text Available A challenge for circulating tumor cell (CTC-based diagnostics is the development of simple and inexpensive methods that reliably detect the diverse cells that make up CTCs. CTC-derived nucleases are one category of proteins that could be exploited to meet this challenge. Advantages of nucleases as CTC biomarkers include: (1 their elevated expression in many cancer cells, including cells implicated in metastasis that have undergone epithelial-to-mesenchymal transition; and (2 their enzymatic activity, which can be exploited for signal amplification in detection methods. Here, we describe a diagnostic assay based on quenched fluorescent nucleic acid probes that detect breast cancer CTCs via their nuclease activity. This assay exhibited robust performance in distinguishing breast cancer patients from healthy controls, and it is rapid, inexpensive, and easy to implement in most clinical labs. Given its broad applicability, this technology has the potential to have a substantive impact on the diagnosis and treatment of many cancers. Keywords: cancer, circulating tumor cells, diagnostic nucleic acids, nucleases, diagnostic markers, breast cancer, liquid biopsy

Solving RNA's structural secrets: interaction with antibodies and crystal structure of a nuclease resistant RNA

International Nuclear Information System (INIS)

Wallace, S.T.

1998-10-01

This Ph.D. thesis concerns the structural characterization of RNA. The work is split into two sections: 1) in vitro selection and characterization of RNAs which bind antibiotics and 2) crystal structure of a nuclease resistant RNA molecule used in antisense applications. Understanding antibiotic-RNA interactions is crucial in aiding rational drug design. We were interested in studying antibiotic interactions with RNAs small enough to characterize at the molecular and possibly at the atomic level. In order to do so, we previously performed in vitro selection to find small RNAs which bind to the peptide antibiotic viomycin and the aminoglycoside antibiotic streptomycin. The characterization of the viomycin-binding RNAs revealed the necessity of a pseudoknot-structure in order to interact with the antibiotic. The RNAs which were selected to interact with streptomycin require the presence of magnesium to bind the antibiotic. One of the RNAs, upon interacting with streptomycin undergoes a significant conformational change spanning the entire RNA sequence needed to bind the antibiotic. In a quest to design oligodeoxynucleotides (ODNs) which are able to specifically bid and inactivate the mRNA of a gene, it is necessary to fulfill two criteria: 1) increase binding affinity between the ODN and the target RNA and 2) increase the ODN's resistance to nuclease degradation. An ODN with an aminopropyl modification at the 2' position of its ribose has emerged as the most successful candidate at fulfilling both criteria. It is the most nuclease resistant modification known to date. We were interested in explaining how this modification is able to circumvent degradation by nucleases. A dodecamer containing a single 2'-O-aminopropyl modified nucleotide was crystallized and the structure was solved to a resolution of 1.6 A. In an attempt to explain the nuclease resistance, the crystal coordinates were modeled into the active exonuclease site of DNA polymerase I. We propose the

Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL

International Nuclear Information System (INIS)

Do, To Uyen; Ho, Bay; Shih, Shyh-Jen; Vaughan, Andrew

2012-01-01

Highlights: ► A Zinc Finger Nuclease (ZFN) targeting a leukemogenic hot spot for rearrangement in MLL is created. ► The novel ZFN efficiently cleaves MLL exon 13. ► Despite MLL cleavage and evidence of mis-repair, no leukemogenic translocations were produced. ► MLL cleavage alone is insufficient to generate leukemogenic translocations. - Abstract: Radiation treatment or chemotherapy has been linked with a higher risk of secondary cancers such as therapy related Acute Myeloid Leukemia (tAML). Several of these cancers have been shown to be correlated to the introduction of double stranded breaks (DSB) and rearrangements within the Mixed Lineage Leukemia (MLL) gene. We used Zinc Finger Nucleases (ZFNs) to introduce precise cuts within MLL to examine how a single DNA DSB might lead to chromosomal rearrangements. A ZFN targeting exon 13 within the Breakpoint Cluster Region of MLL was transiently expressed in a human lymphoblast cell line originating from a CML patient. Although FISH analysis showed ZFN DSB at this region increased the rate of MLL fragmentation, we were unable to detect leukemogenic rearrangements or translocations via inverse PCR. Interestingly, gene fragmentation as well as small interstitial deletions, insertions and base substitutions increased with the inhibition of DNA-PK, suggesting repair of this particular DSB is linked to non-homologous end joining (NHEJ). Although mis-repair of DSBs may be necessary for the initiation of leukemogenic translocations, a MLL targeted DNA break alone is insufficient

Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL

Energy Technology Data Exchange (ETDEWEB)

Do, To Uyen [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Ho, Bay; Shih, Shyh-Jen [Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Vaughan, Andrew, E-mail: Andrew.vaughan@ucdmc.ucdavis.edu [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States)

2012-12-15

Highlights: ► A Zinc Finger Nuclease (ZFN) targeting a leukemogenic hot spot for rearrangement in MLL is created. ► The novel ZFN efficiently cleaves MLL exon 13. ► Despite MLL cleavage and evidence of mis-repair, no leukemogenic translocations were produced. ► MLL cleavage alone is insufficient to generate leukemogenic translocations. - Abstract: Radiation treatment or chemotherapy has been linked with a higher risk of secondary cancers such as therapy related Acute Myeloid Leukemia (tAML). Several of these cancers have been shown to be correlated to the introduction of double stranded breaks (DSB) and rearrangements within the Mixed Lineage Leukemia (MLL) gene. We used Zinc Finger Nucleases (ZFNs) to introduce precise cuts within MLL to examine how a single DNA DSB might lead to chromosomal rearrangements. A ZFN targeting exon 13 within the Breakpoint Cluster Region of MLL was transiently expressed in a human lymphoblast cell line originating from a CML patient. Although FISH analysis showed ZFN DSB at this region increased the rate of MLL fragmentation, we were unable to detect leukemogenic rearrangements or translocations via inverse PCR. Interestingly, gene fragmentation as well as small interstitial deletions, insertions and base substitutions increased with the inhibition of DNA-PK, suggesting repair of this particular DSB is linked to non-homologous end joining (NHEJ). Although mis-repair of DSBs may be necessary for the initiation of leukemogenic translocations, a MLL targeted DNA break alone is insufficient.

Reconstitution of nucleotide excision nuclease with UvrA and UvrB proteins from Escherichia coli and UvrC protein from Bacillus subtilis

International Nuclear Information System (INIS)

Lin, J.J.; Sancar, A.

1990-01-01

Recently, an open reading frame which has a deduced amino acid sequence that shows 38% homology to Escherichia coli UvrC protein was found upstream of the aspartokinase II gene (ask) in Bacillus subtilis. We found that plasmids containing this open reading frame complement the uvrC mutations in E. coli. We joined the open reading frame to a tac promoter to amplify the gene product in E. coli and purified the protein to near homogeneity. The apparent molecular weight of the gene product is 69,000, which is consistent with the calculated molecular weight of 69,378 fro the deduced gene product of the open reading frame. The purified gene product causes the nicking of DNA at the 8th phosphodiester bond 5' and the 5th phosphodiester bond 3' to a thymine dimer when mixed with E. coli UvrA and UvrB proteins and a DNA substrate containing a uniquely located thymine dimer. We conclude that the gene product of the open reading frame is the B. subtilis UvrC protein. Our results suggest that the B. subtilis nucleotide excision repair system is quite similar to that of E. coli. Furthermore, complementation of the UvrA and UvrB proteins from a Gram-negative bacterium with the UvrC protein of Gram-positive B. subtilis indicates a significant evolutionary conservation of the nucleotide excision repair system

CRISPR/Cas9 nuclease-mediated gene knock-in in bovine-induced pluripotent cells.

Science.gov (United States)

Heo, Young Tae; Quan, Xiaoyuan; Xu, Yong Nan; Baek, Soonbong; Choi, Hwan; Kim, Nam-Hyung; Kim, Jongpil

2015-02-01

Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3β and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.

Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption.

Science.gov (United States)

Gopalappa, Ramu; Suresh, Bharathi; Ramakrishna, Suresh; Kim, Hyongbum Henry

2018-03-23

The use of paired Cas9 nickases instead of Cas9 nuclease drastically reduces off-target effects. Because both nickases must function for a nickase pair to make a double-strand break, the efficiency of paired nickases can intuitively be expected to be lower than that of either corresponding nuclease alone. Here, we carefully compared the gene-disrupting efficiency of Cas9 paired nickases with that of nucleases. Interestingly, the T7E1 assay and deep sequencing showed that on-target efficiency of paired D10A Cas9 nickases was frequently comparable, but sometimes higher than that of either corresponding nucleases in mammalian cells. As the underlying mechanism, we found that the HNH domain, which is preserved in the D10A Cas9 nickase, has higher activity than the RuvC domain in mammalian cells. In this study, we showed: (i) the in vivo cleavage efficiency of the HNH domain of Cas9 in mammalian cells is higher than that of the RuvC domain, (ii) paired Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. We envision that our findings which were overlooked in previous reports will serve as a new potential guideline for tool selection for CRISPR-Cas9-mediated gene disruption, facilitating efficient and precise genome editing.

DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

International Nuclear Information System (INIS)

Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D.

1991-01-01

Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links

Recovery from inhibition by UV-irradiation of ornithine decarboxylase induction in human cells: implication of excision repair

Energy Technology Data Exchange (ETDEWEB)

Ben-Hur, E.; Prager, A. (Nuclear Research Centre-Negev, Beer-Sheva (Israel)); Buonaguro, F. (Argonne National Lab., IL (USA))

1982-05-01

Exposure of stationary-phase human breast carcinoma (T-47D) cells to far-UV light (254nm) inhibited the appearance of induced ornithine decarboxylase (ODC) activity. The fluence response curve had a shoulder (Dsub(q)=2Jm/sup -2/) followed by an exponential decline (D/sub 0/=4.2Jm/sup -2/). The cells could recover from this inhibition when the stimulus of induction of ODC was delayed for 20-24h after irradiation. Hydroxyurea (HU) when present at 3mM during the recovery period eliminated completely the ability of the cells to recover. This effect of HU on ODC induction was partially reversed by 50..mu..M of the four deoxyribonucleosides required for DNA synthesis. Neither HU nor the deoxyribonucleosides by themselves affected ODC induction in unirradiated cells. Since HU inhibited the recovery from potentially lethal UV damage and is a known inhibitor of excision repair, it is suggested that recovery from UV-induced inhibition of ODC induction depends on excision-repair of DNA damage. This interpretation is strongly supported by the finding that specific photolysis of 5-bromodeoxyuridine, incorporated into DNA during the recovery period, inhibited recovery of ODC induction from inhibition by UV light.

Crystal structures of two eukaryotic nucleases involved in RNA metabolism

DEFF Research Database (Denmark)

Jonstrup, Anette Thyssen; Midtgaard, Søren Fuglsang; Van, Lan Bich

RNA serves a number of functions in the cell: mRNAs are the carriers of information between gene and protein, tRNAs and rRNAs are involved in the synthesis of proteins, whereas a number of additional RNA species are responsible for other functions in the cell. The quality of the different RNAs...... RNAs. We have solved the structures of two nucleases involved in 3'-5' degradation of RNA; the S. pombe Pop2p and the S. cerevisiae Rrp6p. Pop2p is part of the main cytoplasmatic deadenylation complex in yeast, which also contains the nuclease Ccr4p. Deadenylation, where the poly(A)-tail is removed...... specific transcripts. Here, we present the crystal structure of the S. pombe Pop2p protein to 1.4 Å resolution. The high resolution structure provides a clear picture of the active site architecture. Structural alignment of single nucleotides and poly(A)-oligonucleotides from earlier co-crystal structures...

Antitumor and biological effects of black pine (Pinus nigra) pollen nuclease

Czech Academy of Sciences Publication Activity Database

Lipovová, P.; Podzimek, T.; Orctová, Lidmila; Matoušek, Jaroslav; Poučková, P.; Souček, J.; Matoušek, Josef

2008-01-01

Roč. 55, - (2008), s. 158-164 ISSN 0028-2685 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z50450515 Keywords : pollen nuclease * Antitumor effect Subject RIV: FD - Oncology ; Hematology Impact factor: 1.179, year: 2008

Removal of oxygen free-radical-induced 5′,8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells

Science.gov (United States)

Kuraoka, Isao; Bender, Christina; Romieu, Anthony; Cadet, Jean; Wood, Richard D.; Lindahl, Tomas

2000-01-01

Exposure of cellular DNA to reactive oxygen species generates several classes of base lesions, many of which are removed by the base excision-repair pathway. However, the lesions include purine cyclodeoxynucleoside formation by intramolecular crosslinking between the C-8 position of adenine or guanine and the 5′ position of 2-deoxyribose. This distorting form of DNA damage, in which the purine is attached by two covalent bonds to the sugar-phosphate backbone, occurs as distinct diastereoisomers. It was observed here that both diastereoisomers block primer extension by mammalian and microbial replicative DNA polymerases, using DNA with a site-specific purine cyclodeoxynucleoside residue as template, and consequently appear to be cytotoxic lesions. Plasmid DNA containing either the 5′R or 5′S form of 5′,8-cyclo-2-deoxyadenosine was a substrate for the human nucleotide excision-repair enzyme complex. The R diastereoisomer was more efficiently repaired than the S isomer. No correction of the lesion by direct damage reversal or base excision repair was detected. Dual incision around the lesion depended on the core nucleotide excision-repair protein XPA. In contrast to several other types of oxidative DNA damage, purine cyclodeoxynucleosides are chemically stable and would be expected to accumulate at a slow rate over many years in the DNA of nonregenerating cells from xeroderma pigmentosum patients. High levels of this form of DNA damage might explain the progressive neurodegeneration seen in XPA individuals. PMID:10759556

Alar base reduction: the boomerang-shaped excision.

Science.gov (United States)

Foda, Hossam M T

2011-04-01

A boomerang-shaped alar base excision is described to narrow the nasal base and correct the excessive alar flare. The boomerang excision combined the external alar wedge resection with an internal vestibular floor excision. The internal excision was inclined 30 to 45 degrees laterally to form the inner limb of the boomerang. The study included 46 patients presenting with wide nasal base and excessive alar flaring. All cases were followed for a mean period of 18 months (range, 8 to 36 months). The laterally oriented vestibular floor excision allowed for maximum preservation of the natural curvature of the alar rim where it meets the nostril floor and upon its closure resulted in a considerable medialization of alar lobule, which significantly reduced the amount of alar flare and the amount of external alar excision needed. This external alar excision measured, on average, 3.8 mm (range, 2 to 8 mm), which is significantly less than that needed when a standard vertical internal excision was used ( P boomerang alar base excision proved to be a safe and effective technique for narrowing the nasal base and elimination of the excessive flaring and resulted in a natural, well-proportioned nasal base with no obvious scarring. © Thieme Medical Publishers.

A novel mitochondrial nuclease-associated protein: a major executor of the programmed nuclear death in Tetrahymena thermophila.

Science.gov (United States)

Osada, Eriko; Akematsu, Takahiko; Asano, Tomoya; Endoh, Hiroshi

2014-03-01

Programmed nuclear death (PND) in the ciliate Tetrahymena is an apoptosis-like phenomenon that occurs in a restricted space of cytoplasm during conjugation. In the process, only the parental macronucleus is selectively eliminated from the progeny cytoplasm, in conjunction with differentiation of new macronuclei for the next generation. For the last decade, mitochondria have been elucidated to be a crucial executioner like apoptosis: apoptosis-inducing factor and yet-unidentified nucleases localised in mitochondria are major factors for PND. To identify such nucleases, we performed a DNase assay in a PAGE (SDS-DNA-PAGE) using total mitochondrial proteins. Some proteins showed DNase activity, but particularly a 17 kDa protein exhibited the highest and predominant activity. Mass spectrometric analysis revealed a novel mitochondrial nuclease, named TMN1, whose homologue has been discovered only in the ciliate Paramecium tetraurelia, but not in other eukaryotes. Gene disruption of TMN1 led to a drastic reduction of mitochondrial nuclease activity and blocked nuclear degradation during conjugation, but did not affect accumulation of autophagic and lysosomal machinery around the parental macronucleus. These observations strongly suggest that the mitochondrial nuclease-associated protein plays a key role in PND as a major executor. Taking the novel protein specific to ciliates in consideration, Tetrahymena would have diverted a different protein from common apoptotic factors shared in eukaryotes to PND in the course of ciliate evolution. © 2014 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

Nuclease-like activity of some Cu(II) complexes

International Nuclear Information System (INIS)

Durackova, Z.; Fenikova, L.; Svorenova, L.; Labudova, O.; Kollarova, M.; Labuda, J.

1995-01-01

The nuclease reaction of a copper complex with the macrocyclic Schiff base ligand tetrabenzo[b,f,j,n][a,3,9,13]tetraaza cyclohexadecine (TAAB) at the cleavage of DNA in aerobic conditions and the presence of ascorbic acid has been investigated and compared with that of the copper phenanthroline complex. The AT specifity of the Cu(TAAB) 2+ for both single-stranded and double-stranded DNA templates was observed. (authors), 4 figs., 6 refs

Evaluation of 5 ' nuclease assay for detection of Actinobacillus pleuropneumoniae

DEFF Research Database (Denmark)

Angen, Øystein; Jensen, J.; Lavritsen, D. T.

2001-01-01

Sequence detection by the 5' nuclease TaqMan assay uses online detection of internal fluorogenic probes in closed PCR tubes. Primers and probe were chosen from a part of the omlA gene common to all serotypes of Actinobacillus pleuropneumoniae, which gave an amplicon of 92 bp, The test was evaluat...

Molecular cloning and biological characterization of the human excision repair gene ERCC-3

International Nuclear Information System (INIS)

Weeda, G.; van Ham, R.C.; Masurel, R.; Westerveld, A.; Odijk, H.; de Wit, J.; Bootsma, D.; van der Eb, A.J.; Hoeijmakers, J.H.

1990-01-01

In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion

Structural and Catalytic Properties of S1 Nuclease from Aspergillus oryzae Responsible for Substrate Recognition, Cleavage, Non-Specificity, and Inhibition.

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Tomáš Kovaľ

Full Text Available The single-strand-specific S1 nuclease from Aspergillus oryzae is an archetypal enzyme of the S1-P1 family of nucleases with a widespread use for biochemical analyses of nucleic acids. We present the first X-ray structure of this nuclease along with a thorough analysis of the reaction and inhibition mechanisms and of its properties responsible for identification and binding of ligands. Seven structures of S1 nuclease, six of which are complexes with products and inhibitors, and characterization of catalytic properties of a wild type and mutants reveal unknown attributes of the S1-P1 family. The active site can bind phosphate, nucleosides, and nucleotides in several distinguished ways. The nucleoside binding site accepts bases in two binding modes-shallow and deep. It can also undergo remodeling and so adapt to different ligands. The amino acid residue Asp65 is critical for activity while Asn154 secures interaction with the sugar moiety, and Lys68 is involved in interactions with the phosphate and sugar moieties of ligands. An additional nucleobase binding site was identified on the surface, which explains the absence of the Tyr site known from P1 nuclease. For the first time ternary complexes with ligands enable modeling of ssDNA binding in the active site cleft. Interpretation of the results in the context of the whole S1-P1 nuclease family significantly broadens our knowledge regarding ligand interaction modes and the strategies of adjustment of the enzyme surface and binding sites to achieve particular specificity.

Identity of zinc finger nucleases with specificity to herpes simplex virus type II genomic DNA: novel HSV-2 vaccine/therapy precursors

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Wayengera Misaki

2011-06-01

Full Text Available Abstract Background Herpes simplex type II (HSV-2 is a member of the family herpesviridae. Human infection with this double stranded linear DNA virus causes genital ulcerative disease and existing treatment options only serve to resolve the symptomatology (ulcers associated with active HSV-2 infection but do not eliminate latent virus. As a result, infection with HSV-2 follows a life-long relapsing (active versus latent course. On the basis of a primitive bacterium anti-phage DNA defense, the restriction modification (R-M system, we previously identified the Escherichia coli restriction enzyme (REase EcoRII as a novel peptide to excise or irreversibly disrupt latent HSV-2 DNA from infected cells. However, sequences of the site specificity palindrome of EcoRII 5'-CCWGG-3' (W = A or T are equally present within the human genome and are a potential source of host-genome toxicity. This feature has limited previous HSV-2 EcoRII based therapeutic models to microbicides only, and highlights the need to engineer artificial REases (zinc finger nucleases-ZFNs with specificity to HSV-2 genomic-DNA only. Herein, the therapeutic-potential of zinc finger arrays (ZFAs and ZFNs is identified and modeled, with unique specificity to the HSV-2 genome. Methods and results Using the whole genome of HSV-2 strain HG52 (Dolan A et al.,, and with the ZFN-consortium's CoDA-ZiFiT software pre-set at default, more than 28,000 ZFAs with specificity to HSV-2 DNA were identified. Using computational assembly (through in-silico linkage to the Flavobacterium okeanokoites endonuclease Fok I of the type IIS class, 684 ZFNs with specificity to the HSV-2 genome, were constructed. Graphic-analysis of the HSV-2 genome-cleavage pattern using the afore-identified ZFNs revealed that the highest cleavage-incidence occurred within the 30,950 base-pairs (~between the genomic context coordinates 0.80 and 1.00 at the 3' end of the HSV-2 genome. At approximately 3,095 bp before and after the

Crystallization and preliminary X-ray analysis of a RecB-family nuclease from the archaeon Pyrococcus abyssi

Energy Technology Data Exchange (ETDEWEB)

Ren, Bin, E-mail: ren@csb.ki.se [Center for Structural Biochemistry, Karolinska Institute, NOVUM, S-141 57 Huddinge (Sweden); Kuhn, Joëlle; Meslet-Cladiere, Laurence; Myllykallio, Hannu [Université Paris-Sud, Institut de Génétique et Microbiologie, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8621, F-91405 Orsay CEDEX (France); Ladenstein, Rudolf [Center for Structural Biochemistry, Karolinska Institute, NOVUM, S-141 57 Huddinge (Sweden)

2007-05-01

A RecB-like nuclease from the archaeon Pyrococcus abyssi was expressed, purified and crystallized. The crystals belong to the orthorhombic space group C222{sub 1} with a = 81.5, b = 159.8, c = 100.8 Å, and a native data set was collected to 2.65 Å resolution. Nucleases are required to process and repair DNA damage in living cells. One of the best studied nucleases is the RecB protein, which functions in Escherichia coli as a component of the RecBCD enzyme complex that amends double-strand breaks in DNA. Although archaea do not contain the RecBCD complex, a RecB-like nuclease from Pyrococcus abyssi has been cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant. The crystals belong to the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 81.5, b = 159.8, c = 100.8 Å. Self-rotation function and native Patterson map calculations revealed that there is a dimer in the asymmetric unit with its local twofold axis running parallel to the crystallographic twofold screw axis. The crystals diffracted to about 2 Å and a complete native data set was collected to 2.65 Å resolution.

Erasure of Tet-Oxidized 5-Methylcytosine by a SRAP Nuclease

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Soo-Mi Kweon

2017-10-01

Full Text Available Enzymatic oxidation of 5-methylcytosine (5mC in DNA by the Tet dioxygenases reprograms genome function in embryogenesis and postnatal development. Tet-oxidized derivatives of 5mC such as 5-hydroxymethylcytosine (5hmC act as transient intermediates in DNA demethylation or persist as durable marks, yet how these alternative fates are specified at individual CpGs is not understood. Here, we report that the SOS response-associated peptidase (SRAP domain protein Srap1, the mammalian ortholog of an ancient protein superfamily associated with DNA damage response operons in bacteria, binds to Tet-oxidized forms of 5mC in DNA and catalyzes turnover of these bases to unmodified cytosine by an autopeptidase-coupled nuclease. Biallelic inactivation of murine Srap1 causes embryonic sublethality associated with widespread accumulation of ectopic 5hmC. These findings establish a function for a class of DNA base modification-selective nucleases and position Srap1 as a determinant of 5mC demethylation trajectories during mammalian embryonic development.

Frequency of intrachromosomal homologous recombination induced by UV radiation in normally repairing and excision repair-deficient human cells

International Nuclear Information System (INIS)

Tsujimura, T.; Maher, V.M.; McCormick, J.J.; Godwin, A.R.; Liskay, R.M.

1990-01-01

To investigate the role of DNA damage and nucleotide excision repair in intrachromosomal homologous recombination, a plasmid containing duplicated copies of the gene coding for hygromycin resistance was introduced into the genome of a repair-proficient human cell line, KMST-6, and two repair-deficient lines, XP2OS(SV) from xeroderma pigmentosum complementation group A and XP2YO(SV) from complementation group F. Neither hygromycin-resistance gene codes for a functional enzyme because each contains an insertion/deletion mutation at a unique site, but recombination between the two defective genes can yield hygromycin-resistant cells. The rates of spontaneous recombination in normal and xeroderma pigmentosum cell strains containing the recombination substrate were found to be similar. The frequency of UV-induced recombination was determined for three of these cell strains. At low doses, the group A cell strain and the group F cell strain showed a significant increase in frequency of recombinants. The repair-proficient cell strain required 10-to 20-fold higher doses of UV to exhibit comparable increases in frequency of recombinants. These results suggest that unexcised DNA damage, rather than the excision repair process per se, stimulates such recombination

Medium optimization for nuclease P1 production by Penicillium citrinum in solid-state fermentation using polyurethane foam as inert carrier

NARCIS (Netherlands)

Zhu, Y.; Knol, W.; Smits, J.P.; Bol, J.

1996-01-01

A solid-state fermentation system, using polyurethane foam as an inert carrier, was used for the production of nuclease P1 by Penicillium citrinum. Optimization of nuclease P1 production was carried out using a synthetic liquid medium. After a two-step medium optimization using a fractional

Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.

Science.gov (United States)

Sinkunas, Tomas; Gasiunas, Giedrius; Fremaux, Christophe; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus

2011-04-06

Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.

Multispot array combined with S1 nuclease-mediated elimination of unpaired nucleotides

DEFF Research Database (Denmark)

Yoo, Seung Min; Kim, Dong Min; Lee, Sang Yup

2015-01-01

The accurate detection of mismatched base pairs is critical to many DNA hybridization-based applications in basic research and diagnostics. We herein demonstrate that mismatched DNAs on a multispot array can be accurately detected in a multiplexed way by employing the S1 nuclease-based mismatched...... base pair-specific cleavage system. After the optimization of the reaction condition, mismatched DNAs present in various pathogenic bacteria and genetic disorders could be successfully detected with stable hybridization signals regardless of the position of the fluorescent label relative to the probe......-target duplex. This technique of performing S1 nuclease-mediated cleavage on a multispot array offers high specificity and high-throughput detection of mismatched DNAs. It is expected that this assay system will prove useful for single-assay genotyping and/or the diagnosis of various diseases and pathogens....

Evidence that novobiocin and nalidixic acid do not inhibit excision repair in u.v.-irradiated human skin fibroblasts at a pre-incision

International Nuclear Information System (INIS)

Keyse, S.M.; Tyrrell, R.M.

1985-01-01

The effects of novobiocin and nalidixic acid on the specific toxicity of aphidicolin towards u.v. irradiated arrested human skin fibroblasts have been determined. Contrary to the result expected if either drug were causing inhibition of excision repair at a pre-incision step the sector of toxicity due to a combined treatment of 300 μg ml -1 nalidixic acid and 1.0 μg ml -1 aphidicolin is unchanged when compared with that due to treatment with 1.0 μg ml -1 aphidicolin alone, while that for 150 μg ml -1 novobiocin + 1.0 μg ml -1 aphidicolin was slightly increased. In parallel measurements of the inhibition of u.v.-induced DNA repair synthesis in arrested fibroblasts by these drugs, 150 μg ml -1 novobiocin inhibited repair synthesis by approx.60% over the fluence range employed. Nalidixic acid (300 μg ml -1 ) caused no detectable inhibition of repair synthesis. It was concluded that the mode of action of novobiocin in the inhibition of DNA excision repair is not via the inhibition of a pre-incision step and the data do not support the hypothesis that a type II topoisomerase mediated change in DNA supercoiling is an essential early step in excision repair of u.v.-induced damage. (author)

Modular Nuclease-Responsive DNA Three-Way Junction-Based Dynamic Assembly of a DNA Device and Its Sensing Application.

Science.gov (United States)

Zhu, Jing; Wang, Lei; Xu, Xiaowen; Wei, Haiping; Jiang, Wei

2016-04-05

Here, we explored a modular strategy for rational design of nuclease-responsive three-way junctions (TWJs) and fabricated a dynamic DNA device in a "plug-and-play" fashion. First, inactivated TWJs were designed, which contained three functional domains: the inaccessible toehold and branch migration domains, the specific sites of nucleases, and the auxiliary complementary sequence. The actions of different nucleases on their specific sites in TWJs caused the close proximity of the same toehold and branch migration domains, resulting in the activation of the TWJs and the formation of a universal trigger for the subsequent dynamic assembly. Second, two hairpins (H1 and H2) were introduced, which could coexist in a metastable state, initially to act as the components for the dynamic assembly. Once the trigger initiated the opening of H1 via TWJs-driven strand displacement, the cascade hybridization of hairpins immediately switched on, resulting in the formation of the concatemers of H1/H2 complex appending numerous integrated G-quadruplexes, which were used to obtain label-free signal readout. The inherent modularity of this design allowed us to fabricate a flexible DNA dynamic device and detect multiple nucleases through altering the recognition pattern slightly. Taking uracil-DNA glycosylase and CpG methyltransferase M.SssI as models, we successfully realized the butt joint between the uracil-DNA glycosylase and M.SssI recognition events and the dynamic assembly process. Furthermore, we achieved ultrasensitive assay of nuclease activity and the inhibitor screening. The DNA device proposed here will offer an adaptive and flexible tool for clinical diagnosis and anticancer drug discovery.

Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines

DEFF Research Database (Denmark)

Steentoft, Catharina; Vakhrushev, Sergey Y; Vester-Christensen, Malene B

2011-01-01

Zinc-finger nuclease (ZFN) gene targeting is emerging as a versatile tool for engineering of multiallelic gene deficiencies. A longstanding obstacle for detailed analysis of glycoproteomes has been the extensive heterogeneities in glycan structures and attachment sites. Here we applied ZFN target...

Mung bean sprout (Phaseolus aureus) nuclease and its biological and antitumor effects

Czech Academy of Sciences Publication Activity Database

Souček, J.; Škvor, J.; Poučková, P.; Matoušek, Jaroslav; Slavík, Tomáš; Matoušek, Josef

2006-01-01

Roč. 53, - (2006), s. 402-409 ISSN 0028-2685 R&D Projects: GA ČR GA521/06/1149; GA ČR GA523/04/0755 Keywords : mung bean * nuclease Subject RIV: FD - Oncology ; Hematology Impact factor: 1.247, year: 2006

Evidence that novobiocin and nalidixic acid do not inhibit excision repair in u.v.-irradiated human skin fibroblasts at a pre-incision step

International Nuclear Information System (INIS)

Keyse, S.M.; Tyrrell, R.M.

1985-01-01

The effects of novobiocin and nalidixic acid on the specific toxicity of aphidicolin towards u.v. irradiated arrested (nondividing) human skin fibroblasts have been determined. Contrary to the result expected if either drug were causing inhibition of excision repair at a pre-incision step the sector of toxicity due to a combined treatment of 300 micrograms ml -1 nalidixic acid and 1.0 micrograms ml -1 aphidicolin is unchanged when compared with that due to treatment with 1.0 micrograms ml -1 aphidicolin alone, while that for 150 micrograms ml -1 novobiocin + 1.0 micrograms ml -1 aphidicolin was slightly increased. In parallel measurements of the inhibition of u.v.-induced DNA repair synthesis in arrested fibroblasts by these drugs, 150 micrograms ml -1 novobiocin inhibited repair synthesis by approximately 60% over the fluence range employed. Nalidixic acid at a concentration of 300 micrograms ml -1 caused no detectable inhibition of repair synthesis. The authors conclude that the mode of action of novobiocin in the inhibition of DNA excision repair is not via the inhibition of a pre-incision step and the data do not support the hypothesis that a type II topoisomerase mediated change in DNA supercoiling is an essential early step in excision repair of u.v.-induced damage

Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease.

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Megan R Kiedrowski

Full Text Available Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2 that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme.

Multidirectional Vector Excision Leads to Better Outcomes than Traditional Elliptical Excision of Facial Congenital Melanocytic Nevus

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Seung Il Oh

2013-09-01

Full Text Available Background The elliptical excision is the standard method of removing benign skin lesions,such as congenital melanocytic nevi. This technique allows for primary closure, with little to nodog-ear deformity, but may sacrifice normal tissue adjacent to the lesion, resulting in scarswhich are unnecessarily long. This study was designed to compare the predicted results ofelliptical excision with those resulting from our excision technique.Methods Eighty-two patients with congenital melanocytic nevus on the face were prospectivelystudied. Each lesion was examined and an optimal ellipse was designed and marked onthe skin. After an incision on one side of the nevus margin, subcutaneous undermining wasperformed in the appropriate direction. The skin flap was pulled up and approximated alongseveral vectors to minimize the occurrence of dog-ear deformity.Results Overall, the final wound length was 21.1% shorter than that achieved by ellipticalexcision. Only 8.5% of the patients required dog-ear repair. There was no significant distortionof critical facial structures. All of the scars were deemed aesthetically acceptable based ontheir Patient and Observer Scar Assessment Scale scores.Conclusions When compared to elliptical excision, our technique appears to minimize dogeardeformity and decrease the final wound length. This technique should be considered analternative method for excision of facial nevi.

Excision of thymine dimers from specifically incised DNA by extracts of xeroderma pigmentosum cells

Energy Technology Data Exchange (ETDEWEB)

Cook, K; Friedberg, E C; Slor, H; Cleaver, J E

1975-07-17

DNA repair defects as exhibited in fibroblasts from patients with xeroderma pigmentosa were studied. Five complementation groups for excision-repair defects were examined to test the hypothesis that a defective endonuclease or exonuclease may be the cause. No evidence was found to indicate that the enzyme activity functions in dimer excision. Since ultraviolet irradiated E. coli DNA incised with an endonuclease purified from phage-infected cells were used, it is possible that other factors may be involved in human UV endonuclease action. (JWP)

Crystallization and preliminary X-ray characterization of two thermostable DNA nucleases

International Nuclear Information System (INIS)

Kuettner, E. Bartholomeus; Pfeifer, Sven; Keim, Antje; Greiner-Stöffele, Thomas; Sträter, Norbert

2006-01-01

Two thermostable DNA nucleases from archaea were crystallized in different space groups; the crystals were suitable for X-ray analysis. Temperature-tolerant organisms are an important source to enhance the stability of enzymes used in biotechnological processes. The DNA-cleaving enzyme exonuclease III from Escherichia coli is used in several applications in gene technology. A thermostable variant could expand the applicability of the enzyme in these methods. Two homologous nucleases from Archaeoglobus fulgidus (ExoAf) and Methanothermobacter thermoautrophicus (ExoMt) were studied for this purpose. Both enzymes were crystallized in different space groups using (poly)ethylene glycols, 2,4-methyl pentandiol, dioxane, ethanol or 2-propanol as precipitants. The addition of a 10-mer DNA oligonucleotide was important to obtain monoclinic crystals of ExoAf and ExoMt that diffracted to resolutions better than 2 Å using synchrotron radiation. The crystal structures of the homologous proteins can serve as templates for genetic engineering of the E. coli exonuclease III and will aid in understanding the different catalytic properties of the enzymes

Genome editing in mouse spermatogonial stem/progenitor cells using engineered nucleases.

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Danielle A Fanslow

Full Text Available Editing the genome to create specific sequence modifications is a powerful way to study gene function and promises future applicability to gene therapy. Creation of precise modifications requires homologous recombination, a very rare event in most cell types that can be stimulated by introducing a double strand break near the target sequence. One method to create a double strand break in a particular sequence is with a custom designed nuclease. We used engineered nucleases to stimulate homologous recombination to correct a mutant gene in mouse "GS" (germline stem cells, testicular derived cell cultures containing spermatogonial stem cells and progenitor cells. We demonstrated that gene-corrected cells maintained several properties of spermatogonial stem/progenitor cells including the ability to colonize following testicular transplantation. This proof of concept for genome editing in GS cells impacts both cell therapy and basic research given the potential for GS cells to be propagated in vitro, contribute to the germline in vivo following testicular transplantation or become reprogrammed to pluripotency in vitro.

Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

Science.gov (United States)

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

2015-07-23

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

Automated 5 ' nuclease assay for detection of virulence factors in porcine Escherichia coli

DEFF Research Database (Denmark)

Frydendahl, K.; Imberechts, H.; Lehmann, S.

2001-01-01

(STa, STb, EAST1) and heat labile LT) enterotoxins and the verocytotoxin variant 2e (VT2e). To correctly identify false negative results, an endogenous internal control targeting the E. coil 16S rRNA gene was incorporated in each test tube. The assay was evaluated using a collection of E. coil...... reference strains which have previously been examined with phenotypical assays or DNA hybridization. Furthermore, the assay was evaluated by testing porcine E. coil field strains, previously characterized. The 5' nuclease assay correctly detected the presence of virulence genes in all reference strains....... When testing field strains there was generally excellent agreement with results obtained by laboratories in Belgium and Germany. In conclusion, the 5' nuclease assay developed is a fast and specific tool for detection of E. coli virulence genes in the veterinary diagnostic laboratory....

Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme

Czech Academy of Sciences Publication Activity Database

Stránský, J.; Koval, Tomáš; Podzimek, T.; Týcová, A.; Lipovová, P.; Matoušek, J.; Kolenko, Petr; Fejfarová, Karla; Dušková, J.; Skálová, T.; Hašek, J.; Dohnálek, Jan

2015-01-01

Roč. 71, č. 11 (2015), s. 1408-1415 ISSN 2053-230X R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) EE2.3.30.0029 Institutional support: RVO:61389013 Keywords : tomato multifunctional nuclease * TBN1 * type I nuclease Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.647, year: 2015

Excision without excision

International Nuclear Information System (INIS)

Brown, David; Sarbach, Olivier; Schnetter, Erik; Diener, Peter; Tiglio, Manuel; Hawke, Ian; Pollney, Denis

2007-01-01

to turducken (turduckens, turduckening, turduckened, turduckened) [math.]: To stuff a black hole. We analyze and apply an alternative to black hole excision based on smoothing the interior of black holes with arbitrary initial data, and solving the vacuum Einstein evolution equations everywhere. By deriving the constraint propagation system for our hyperbolic formulation of the BSSN evolution system we rigorously prove that the constraints propagate causally and so any constraint violations introduced inside the black holes cannot affect the exterior spacetime. We present evolutions of Cook-Pfeiffer binary black hole initial configurations showing that these techniques appear to work robustly for generic data. We also present evidence from spherically symmetric evolutions that for the gauge conditions used the same stationary end-state is approached irrespective of the choice of initial data and smoothing procedure

A Biochemical Approach to Understanding the Fanconi Anemia Pathway-Regulated Nucleases in Genome Maintenance for Preventing Bone Marrow Failure and Cancer

Science.gov (United States)

2014-04-01

the Fanconi Anemia Pathway- Regulated Nucleases in Genome Maintenance for Preventing Bone Marrow Failure and Cancer PRINCIPAL INVESTIGATOR...GRANT NUMBER 4. TITLE AND SUBTITLE A Biochemical Approach to Understanding the Fanconi Anemia Pathway-Regulated Nucleases in Genome Maintenance for...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Fanconi anemia is the most prevalent inherited BMF syndromes, caused by mutations in

Surgical excision of eroded mesh after prior abdominal sacrocolpopexy.

Science.gov (United States)

South, Mary M T; Foster, Raymond T; Webster, George D; Weidner, Alison C; Amundsen, Cindy L

2007-12-01

We previously described an endoscopic-assisted transvaginal mesh excision technique. This study compares surgical outcomes after transvaginal mesh excision vs endoscopic-assisted transvaginal mesh excision. In addition, we reviewed our postoperative outcomes with excision via laparotomy. This was an inclusive retrospective analysis of patients presenting to our institution from 1997 to 2006 for surgical management of vaginal erosion of permanent mesh after sacrocolpopexy. Three techniques were utilized: transvaginal, endoscopic-assisted transvaginal, and laparotomy. For the patients undergoing transvaginal excision, data recorded included number and type of excisions performed, number of prior excisions performed at outside facilities, intraoperative and postoperative complications (including blood transfusions, pelvic abscess, or bowel complications), use of postoperative antibiotics, persistent symptoms of vaginal bleeding and discharge at follow-up, and demographic characteristics. The intraoperative and postoperative complications and the postoperative symptoms were recorded for the laparotomy cases. Thirty-one patients underwent transvaginal mesh excision during this time period: 17 endoscopic-assisted transvaginal and 14 transvaginal without endoscope assistance. In addition, a total of 7 patients underwent abdominal excision via laparotomy. Comparison of the 2 vaginal methods revealed no difference in the demographics or success rate, with success defined as no symptoms at follow-up. Endoscopic-assisted transvaginal excision was successful in 7 of 17 patients and transvaginal without endoscopic assistance in 9 of 13 patients (1 patient excluded for lack of follow-up data) for a total vaginal success rate of 53.3%. No intraoperative and only minor postoperative complications occurred with either vaginal method. Three patients underwent 3 vaginal attempts to achieve complete symptom resolution. The average follow-up time for the entire vaginal group was 14

A 'new lease of life': FnCpf1 possesses DNA cleavage activity for genome editing in human cells.

Science.gov (United States)

Tu, Mengjun; Lin, Li; Cheng, Yilu; He, Xiubin; Sun, Huihui; Xie, Haihua; Fu, Junhao; Liu, Changbao; Li, Jin; Chen, Ding; Xi, Haitao; Xue, Dongyu; Liu, Qi; Zhao, Junzhao; Gao, Caixia; Song, Zongming; Qu, Jia; Gu, Feng

2017-11-02

Cpf1 nucleases were recently reported to be highly specific and programmable nucleases with efficiencies comparable to those of SpCas9. AsCpf1 and LbCpf1 require a single crRNA and recognize a 5'-TTTN-3' protospacer adjacent motif (PAM) at the 5' end of the protospacer for genome editing. For widespread application in precision site-specific human genome editing, the range of sequences that AsCpf1 and LbCpf1 can recognize is limited due to the size of this PAM. To address this limitation, we sought to identify a novel Cpf1 nuclease with simpler PAM requirements. Specifically, here we sought to test and engineer FnCpf1, one reported Cpf1 nuclease (FnCpf1) only requires 5'-TTN-3' as a PAM but does not exhibit detectable levels of nuclease-induced indels at certain locus in human cells. Surprisingly, we found that FnCpf1 possesses DNA cleavage activity in human cells at multiple loci. We also comprehensively and quantitatively examined various FnCpf1 parameters in human cells, including spacer sequence, direct repeat sequence and the PAM sequence. Our study identifies FnCpf1 as a new member of the Cpf1 family for human genome editing with distinctive characteristics, which shows promise as a genome editing tool with the potential for both research and therapeutic applications. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mapping of gene transcripts by nuclease protection assays and cDNA primer extension

International Nuclear Information System (INIS)

Calzone, F.J.; Britten, R.J.; Davidson, E.J.

1987-01-01

An important problem often faced in the molecular characterization of genes is the precise mapping of those genomic sequences transcribed into RNA. This requires identification of the genomic site initiating gene transcription, the location of genomic sequences removed from the primary gene transcript during RNA processing, and knowledge of sequences terminating the processed gene transcript. The objective of the protocols described here is the generation of transcription maps utilizing relatively uncharacterized gene fragments. The basic approach is hybridization of a single-stranded DNA probe with cellular RNA, followed by treatment with a single-strand-specific nuclease that does not attack DNA-RNA hybrids, in order to destroy any unreacted probe sequences. Thus the probe sequences included in the hybrid duplexes are protected from nuclease digestion. The sizes of the protected probe fragments determined by gel electrophoresis correspond to the lengths of the hybridized sequence elements

Low Prevalence of Oral and Nasal Human Papillomavirus in Employees Performing CO2-laser Evaporation of Genital Warts or Loop Electrode Excision Procedure of Cervical Dysplasia

DEFF Research Database (Denmark)

Kofoed, Kristian; Norrbom, Christina; Forslund, Ola

2014-01-01

Risk of human papillomavirus (HPV) transmission during laser vaporisation of genital warts or loop electrode excision procedure is controversial. An oral rinse, a nasal swabs, history of HPV related diseases and data on HPV exposure were collected from 287 employees at departments of dermato......, or loop electrode excision procedure compared with those who did not. HPV 6 or 11 were not detected in any samples. Hand warts after the age of 24 years was more common among dermatology than among non-dermatology personnel (18% vs. 8.0%, p = 0.03). Mucosal HPV types are infrequent in the oral and nasal...... cavity of health care personnel, however, employees at departments of dermato-venereology are at risk of acquiring hand warts....

29 CFR 779.264 - Excise taxes separately stated.

Science.gov (United States)

2010-07-01

... AS APPLIED TO RETAILERS OF GOODS OR SERVICES Employment to Which the Act May Apply; Enterprise Coverage Excise Taxes § 779.264 Excise taxes separately stated. A tax is separately stated where it clearly... 29 Labor 3 2010-07-01 2010-07-01 false Excise taxes separately stated. 779.264 Section 779.264...

Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme

Czech Academy of Sciences Publication Activity Database

Stránský, Jan; Koval, Tomáš; Podzimek, T.; Týcová, Anna; Lipovová, P.; Matoušek, Jaroslav; Kolenko, Petr; Fejfarová, Karla; Dušková, Jarmila; Skálová, Tereza; Hašek, Jindřich; Dohnálek, Jan

2015-01-01

Roč. 71, č. 11 (2015), s. 1408-1415 ISSN 2053-230X R&D Projects: GA MŠk LG14009; GA MŠk(CZ) EE2.3.30.0029; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 ; RVO:60077344 Keywords : tomato multifunctional nuclease * TBN1 * type I nuclease * superhelix Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.647, year: 2015

Staphylococcal nuclease active-site amino acids: pH dependence of tyrosines and arginines by 13C NMR and correlation with kinetic studies

International Nuclear Information System (INIS)

Grissom, C.G.; Markley, J.L.

1989-01-01

The pH and temperature dependence of the kinetic parameters of staphylococcal nuclease have been examined with three p-nitrophenyl phosphate containing DNA analogues that vary as to 3'-substituent. With wild-type (Foggi variant) nuclease (nuclease wt) and the substrates thymidine 3'-phosphate 5'-(p-nitrophenyl phosphate) (PNPdTp), thymidine 3'-methylphosphonate 5'-(p-nitrophenyl phosphate) (PNPdTp Me), and thymidine 5'-(p-nitrophenyl phosphate) (PNPdT), k cat remains nearly constant at 13 min -1 . However, k cat /k m with nuclease wt varies considerably. The data suggests that the inflection k cat /K m with pK a at 9.67 arises from ionization of tyrosine-85, which hydrogen bonds to the divalent 3'-phosphomonester of substrates with this substituent. The enthalpy of ionization of both deprotonation steps in the k cat /K m versus pH profile is 5 kcal/mol. 13 C NMR has been used to determine the pK a values of the arginine and tyrosine residues. The results do not rule out arginine as a candidate for the acidic catalyst that protonates the 5'-ribose alkoxide prior to product release. The phenolic hydroxyl carbon of tyrosine-85 has been assigned by comparing the 13 C NMR spectrum of nuclease wt and nuclease Y85F. This correlation between pK a values along with the absence of other candidates indicates that the ionization of tyrosine-85 is the pK a seen in the k cat /K m vs pH profile for substrates with a divalent 3'-phosphomonester. This conclusion is consistent with the proposed role of tyrosine-85 as a hydrogen-bond donor to the 3'-phosphomonoester of substrates poised for exonucleolytic hydrolysis

A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells

Science.gov (United States)

Tu, Mengjun; Lin, Li; Cheng, Yilu; He, Xiubin; Sun, Huihui; Xie, Haihua; Fu, Junhao; Liu, Changbao; Li, Jin; Chen, Ding; Xi, Haitao; Xue, Dongyu; Liu, Qi; Zhao, Junzhao; Gao, Caixia; Song, Zongming; Qu, Jia

2017-01-01

Abstract Cpf1 nucleases were recently reported to be highly specific and programmable nucleases with efficiencies comparable to those of SpCas9. AsCpf1 and LbCpf1 require a single crRNA and recognize a 5′-TTTN-3′ protospacer adjacent motif (PAM) at the 5′ end of the protospacer for genome editing. For widespread application in precision site-specific human genome editing, the range of sequences that AsCpf1 and LbCpf1 can recognize is limited due to the size of this PAM. To address this limitation, we sought to identify a novel Cpf1 nuclease with simpler PAM requirements. Specifically, here we sought to test and engineer FnCpf1, one reported Cpf1 nuclease (FnCpf1) only requires 5′-TTN-3′ as a PAM but does not exhibit detectable levels of nuclease-induced indels at certain locus in human cells. Surprisingly, we found that FnCpf1 possesses DNA cleavage activity in human cells at multiple loci. We also comprehensively and quantitatively examined various FnCpf1 parameters in human cells, including spacer sequence, direct repeat sequence and the PAM sequence. Our study identifies FnCpf1 as a new member of the Cpf1 family for human genome editing with distinctive characteristics, which shows promise as a genome editing tool with the potential for both research and therapeutic applications. PMID:28977650

Terahertz pulsed imaging of freshly excised human colonic tissues

Energy Technology Data Exchange (ETDEWEB)

Reid, Caroline B; Gibson, Adam P [Department of Medical Physics and Bioengineering, University College London, London, WC1E 6BT (United Kingdom); Fitzgerald, Anthony; Wallace, Vincent P [School of Physics, University of Western Australia, Crawley 6009 (Australia); Reese, George; Tekkis, Paris [Division of Surgery, Chelsea and Westminster Campus, Imperial College London, London (United Kingdom); Goldin, Robert [Centre for Pathology, Imperial College London, St Mary' s Campus, London (United Kingdom); O' Kelly, P S [TeraView Ltd, Platinum Building, St John' s Innovation Park, Cowley Road, Cambridge, CB4 0WS (United Kingdom); Pickwell-MacPherson, Emma, E-mail: c.reid@medphys.ucl.ac.uk [Department of Electronic Engineering, Chinese University of Hong Kong, Shatin, NT (Hong Kong)

2011-07-21

We present the results from a feasibility study which measures properties in the terahertz frequency range of excised cancerous, dysplastic and healthy colonic tissues from 30 patients. We compare their absorption and refractive index spectra to identify trends which may enable different tissue types to be distinguished. In addition, we present statistical models based on variations between up to 17 parameters calculated from the reflected time and frequency domain signals of all the measured tissues. These models produce a sensitivity of 82% and a specificity of 77% in distinguishing between healthy and all diseased tissues and a sensitivity of 89% and a specificity of 71% in distinguishing between dysplastic and healthy tissues. The contrast between the tissue types was supported by histological staining studies which showed an increased vascularity in regions of increased terahertz absorption.

Terahertz pulsed imaging of freshly excised human colonic tissues

International Nuclear Information System (INIS)

Reid, Caroline B; Gibson, Adam P; Fitzgerald, Anthony; Wallace, Vincent P; Reese, George; Tekkis, Paris; Goldin, Robert; O'Kelly, P S; Pickwell-MacPherson, Emma

2011-01-01

We present the results from a feasibility study which measures properties in the terahertz frequency range of excised cancerous, dysplastic and healthy colonic tissues from 30 patients. We compare their absorption and refractive index spectra to identify trends which may enable different tissue types to be distinguished. In addition, we present statistical models based on variations between up to 17 parameters calculated from the reflected time and frequency domain signals of all the measured tissues. These models produce a sensitivity of 82% and a specificity of 77% in distinguishing between healthy and all diseased tissues and a sensitivity of 89% and a specificity of 71% in distinguishing between dysplastic and healthy tissues. The contrast between the tissue types was supported by histological staining studies which showed an increased vascularity in regions of increased terahertz absorption.

Oxidative Damage to RPA Limits the Nucleotide Excision Repair Capacity of Human Cells.

Science.gov (United States)

Guven, Melisa; Brem, Reto; Macpherson, Peter; Peacock, Matthew; Karran, Peter

2015-11-01

Nucleotide excision repair (NER) protects against sunlight-induced skin cancer. Defective NER is associated with photosensitivity and a high skin cancer incidence. Some clinical treatments that cause photosensitivity can also increase skin cancer risk. Among these, the immunosuppressant azathioprine and the fluoroquinolone antibiotics ciprofloxacin and ofloxacin interact with UVA radiation to generate reactive oxygen species that diminish NER capacity by causing protein damage. The replication protein A (RPA) DNA-binding protein has a pivotal role in DNA metabolism and is an essential component of NER. The relationship between protein oxidation and NER inhibition was investigated in cultured human cells expressing different levels of RPA. We show here that RPA is limiting for NER and that oxidative damage to RPA compromises NER capability. Our findings reveal that cellular RPA is surprisingly vulnerable to oxidation, and we identify oxidized forms of RPA that are associated with impaired NER. The vulnerability of NER to inhibition by oxidation provides a connection between cutaneous photosensitivity, protein damage, and increased skin cancer risk. Our findings emphasize that damage to DNA repair proteins, as well as to DNA itself, is likely to be an important contributor to skin cancer risk.

TALEN- and CRISPR/Cas9-Mediated Gene Editing in Human Pluripotent Stem Cells Using Lipid-Based Transfection.

Science.gov (United States)

Hendriks, William T; Jiang, Xin; Daheron, Laurence; Cowan, Chad A

2015-08-03

Using custom-engineered nuclease-mediated genome editing, such as Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA-guided Cas9 nucleases, human pluripotent stem cell (hPSC) lines with knockout or mutant alleles can be generated and differentiated into various cell types. This strategy of genome engineering in hPSCs will prove invaluable for studying human biology and disease. Here, we provide a detailed protocol for design and construction of TALEN and CRISPR vectors, testing of their nuclease activity, and delivery of TALEN or CRISPR vectors into hPSCs. In addition, we describe the use of single-stranded oligodeoxynucleotides (ssODNs) to introduce or repair point mutations. Next, we describe the identification of edited hPSC clones without antibiotic selection, including their clonal selection, genotyping, and expansion for downstream applications. Copyright © 2015 John Wiley & Sons, Inc.

Metal inhibition of human alkylpurine-DNA-N-glycosylase activityin base excision repair

Energy Technology Data Exchange (ETDEWEB)

Wang, Ping; Guliaev, Anton B.; Hang, Bo

2006-02-28

Cadmium (Cd{sup 2+}), nickel (Ni{sup 2+}) and cobalt (Co{sup 2+}) are human and/or animal carcinogens. Zinc (Zn{sup 2+}) is not categorized as a carcinogen, and rather an essential element to humans. Metals were recently shown to inhibit DNA repair proteins that use metals for their function and/or structure. Here we report that the divalent ions Cd{sup 2+}, Ni{sup 2+}, and Zn{sup 2+} can inhibit the activity of a recombinant human N-methylpurine-DNA glycosylase (MPG) toward a deoxyoligonucleotide with ethenoadenine (var epsilonA). MPG removes a variety of toxic/mutagenic alkylated bases and does not require metal for its catalytic activity or structural integrity. At concentrations starting from 50 to 1000 {micro}M, both Cd{sup 2+} and Zn{sup 2+} showed metal-dependent inhibition of the MPG catalytic activity. Ni{sup 2+} also inhibited MPG, but to a lesser extent. Such an effect can be reversed with EDTA addition. In contrast, Co{sup 2+} and Mg{sup 2+} did not inhibit the MPG activity in the same dose range. Experiments using HeLa cell-free extracts demonstrated similar patterns of inactivation of the var epsilonA excision activity by the same metals. Binding of MPG to the substrate was not significantly affected by Cd{sup 2+}, Zn{sup 2+}, and Ni{sup 2+} at concentrations that show strong inhibition of the catalytic function, suggesting that the reduced catalytic activity is not due to altered MPG binding affinity to the substrate. Molecular dynamics (MD) simulations with Zn{sup 2+} showed that the MPG active site has a potential binding site for Zn{sup 2+}, formed by several catalytically important and conserved residues. Metal binding to such a site is expected to interfere with the catalytic mechanism of this protein. These data suggest that inhibition of MPG activity may contribute to metal genotoxicity and depressed repair of alkylation damage by metals in vivo.

Value of histopathologic analysis of subcutis excisions by general practitioners

Directory of Open Access Journals (Sweden)

Verweij Wim

2007-01-01

Full Text Available Abstract Background Only around 60% of skin lesions excised by GPs are referred to a pathologist. Clinical diagnoses of skin excisions by GPs may not be very accurate. Subcutis excisions are rarely done by GPs, and there is hence little information in the literature on the histopathological yield of subcutis excisions by GPs with regard to malignancies. The aim of this study was to evaluate the yield of histopathological investigation of a relatively large group of subcutis excisions by GPs, with special emphasis on discrepancies between clinical and histopathological diagnoses of malignancy. Methods We investigated a series of 90 subcutis excisions, which was derived from a database of consecutive GP submissions from the years 1999–2000 where in the same time period 4595 skin excisions were performed by the same group of GPs. This underlines the apparent reluctance of GPs to perform subcutis excisions. Results The final diagnosis was benign in 88 cases (97.8% and malignant in 2 cases (2.2%. Seven cases had no clinical diagnosis, all of which were benign. Of the 83 clinically benign cases, 81 (97.6% were indeed benign and 2 (2.4% were malignant: one Merkel cell carcinoma and one dermatofibrosarcoma protuberans. The former was clinically thought to be a lipoma, and the latter a trichilemmal cyst. The dermatofibrosarcoma protuberans presented at the age of 27, and the Merkel cell carcinoma at the age of 60. Both were incompletely removed and required re-excision by a surgical oncologist. Conclusion Histopathological investigation of subcutis excisions by GPs yields unexpected and rare malignancies in about 2% of cases that may initially be excised inadequately. Based on these data, and because of the relatively rareness of these type of excisions, it could be argued that it may be worthwhile to have all subcutis excisions by GPs routinely investigated by histopathology.

Activity of some nucleases of cotton sorts and species of various radiosensitivity

International Nuclear Information System (INIS)

Nazirov, N.N.; Arslanova, S.B.

1979-01-01

The activity of some nucleases under the effect of gamma rays was studied on cotton varieties and species differing in radiosensitivity. It was found that acid nuclease was more active in wild cotton forms as compared to the cultivated varieties, whereas with alkaline DNA-ase it was opposite. At the radiation dose of 30 kR the activity of alkaline DNA-ase activated in 26-chromosome wild cotton G. arboreum ssp. alfusifalium and 52-chromosome S.h.ssp.mexicanum, while the activity of acid DNA-ase somewhat decreased. Under irradiating AN-402 variety (produced from ssp. mexicanum by irradiation) the activity of alkaline DNA-ase increased noticeably when budding, whereas the activity of acid DNA-ase was at the level of control. The activity of the alkaline DNA-ase form normalized in the phase of blooming. In C-70-59 variety (G.arboreum) the activity of both DNA-ases increased after irradiation in the phase of blooming. The activity of acid DNA-ase and RNA-ase drastically activated in guza 183 (G. herbaceum) under gamma irradiation, whereas that of alkaline ones remained unchanged

Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease

Science.gov (United States)

Flowers, G. Parker; Timberlake, Andrew T.; Mclean, Kaitlin C.; Monaghan, James R.; Crews, Craig M.

2014-01-01

Among tetrapods, only urodele salamanders, such as the axolotl Ambystoma mexicanum, can completely regenerate limbs as adults. The mystery of why salamanders, but not other animals, possess this ability has for generations captivated scientists seeking to induce this phenomenon in other vertebrates. Although many recent advances in molecular biology have allowed limb regeneration and tissue repair in the axolotl to be investigated in increasing detail, the molecular toolkit for the study of this process has been limited. Here, we report that the CRISPR-Cas9 RNA-guided nuclease system can efficiently create mutations at targeted sites within the axolotl genome. We identify individual animals treated with RNA-guided nucleases that have mutation frequencies close to 100% at targeted sites. We employ this technique to completely functionally ablate EGFP expression in transgenic animals and recapitulate developmental phenotypes produced by loss of the conserved gene brachyury. Thus, this advance allows a reverse genetic approach in the axolotl and will undoubtedly provide invaluable insight into the mechanisms of salamanders' unique regenerative ability. PMID:24764077

76 FR 52862 - Time for Payment of Certain Excise Taxes, and Quarterly Excise Tax Payments for Small Alcohol...

Science.gov (United States)

2011-08-24

... 40 Cigars and cigarettes, Claims, Electronic fund transfers, Excise taxes, Labeling, Packaging and... that are not required to pay taxes through electronic funds transfer (EFT), this first payment period..., Electronic funds transfers, Excise taxes, Exports, Food additives, Fruit juices, Labeling, Liquors, Packaging...

Dynamics and denaturation of a protein. Simulations and neutron scattering on staphylococcus nuclease

International Nuclear Information System (INIS)

Goupil-Lamy, Anne

1997-01-01

This research thesis reports simulations and experiments of inelastic scattering on the whole frequency spectrum to analyse the vibrations of the staphylococcus nuclease and its fragment, in order to study protein folding. Based on these experiments, information on eigenvectors which describe vibration modes can be directly obtained. Inelastic intensities are indeed fully determined by nuclear cross sections and the mean square displacement of each atom. Some experimentally noticed peaks are then explained by calculating a theoretical spectrum from an analysis of normal modes. The studied fragment is made of 136 c-terminal residues. The fragment structure obtained by molecular dynamics simulation is compared with available experimental data. Then, experiments of neutron scattering on the nuclease of staphylococcus and its fragment have been performed. Quasi elastic scattering spectra have been measured. The author then used simulations to try to reproduce the quasi-elastic spectrum. Experiments of inelastic scattering have then been performed [fr

Response surface optimization of carbon and nitrogen sources for nuclease P1 production by Penicillium citrinum F-5-5

International Nuclear Information System (INIS)

Liang Xinle; Huang Yingying; Zhang Hong; Chen Min; Liu Xuan

2011-01-01

Penicillium citrinum F-5-5, a nuclease P1 high-producing strain with 978.6 U/ml in potato glucose medium, was derived from the original Penicillium citrinum CICC 4011 with 60 Co γ-rays irradiation mutation and then protoplasts fusion treatment. Culture components were optimized for the nuclease P1 production, and response surface methodology was applied for the critical medium components(carbon and nitrogen sources) which were preselected by Plackett-Burman design approach. Glucose, soluble starch and corn steep powder showed significant effects on production of nuclease. Central composite design was used for the optimization levels by software Minitab 15, and it showed that, the optimal values for the concentration of glucose, soluble starch and corn steep powder were 30.89, 42.46 and 11.60 g/L, respectively. With this medium,an enzyme activity of 1687.16 U/ml could be obtained theoretically. Using this optimized medium, an experimental enzyme activity of 1672.6 U/ml was reached. (authors)

An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease.

Science.gov (United States)

Wiemels, Richard E; Cech, Stephanie M; Meyer, Nikki M; Burke, Caleb A; Weiss, Andy; Parks, Anastacia R; Shaw, Lindsey N; Carroll, Ronan K

2017-01-01

Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently

Circumareolar Incision‑subdermal Tunneling Dissection for Excision ...

African Journals Online (AJOL)

2017 Nigerian Journal of Surgery | Published by Wolters Kluwer - Medknow. Excision of ... This is a report of excision of MF in multiple quadrants of the breast using a ... Agodirin, et al. .... the breast: The Ribeiro technique modified by Rezai.

Nasal base narrowing: the combined alar base excision technique.

Science.gov (United States)

Foda, Hossam M T

2007-01-01

To evaluate the role of the combined alar base excision technique in narrowing the nasal base and correcting excessive alar flare. The study included 60 cases presenting with a wide nasal base and excessive alar flaring. The surgical procedure combined an external alar wedge resection with an internal vestibular floor excision. All cases were followed up for a mean of 32 (range, 12-144) months. Nasal tip modification and correction of any preexisting caudal septal deformities were always completed before the nasal base narrowing. The mean width of the external alar wedge excised was 7.2 (range, 4-11) mm, whereas the mean width of the sill excision was 3.1 (range, 2-7) mm. Completing the internal excision first resulted in a more conservative external resection, thus avoiding any blunting of the alar-facial crease. No cases of postoperative bleeding, infection, or keloid formation were encountered, and the external alar wedge excision healed with an inconspicuous scar that was well hidden in the depth of the alar-facial crease. Finally, the risk of notching of the alar rim, which can occur at the junction of the external and internal excisions, was significantly reduced by adopting a 2-layered closure of the vestibular floor (P = .01). The combined alar base excision resulted in effective narrowing of the nasal base with elimination of excessive alar flare. Commonly feared complications, such as blunting of the alar-facial crease or notching of the alar rim, were avoided by using simple modifications in the technique of excision and closure.

75 FR 9359 - Drawback of Internal Revenue Excise Tax

Science.gov (United States)

2010-03-02

... Drawback of Internal Revenue Excise Tax AGENCY: Customs and Border Protection, Department of Homeland... substitution drawback claim for internal revenue excise tax paid on imported merchandise in situations where no excise tax was paid upon the substituted merchandise or where the substituted merchandise is the subject...

29 CFR 779.262 - Excise taxes at the retail level.

Science.gov (United States)

2010-07-01

... AS APPLIED TO RETAILERS OF GOODS OR SERVICES Employment to Which the Act May Apply; Enterprise Coverage Excise Taxes § 779.262 Excise taxes at the retail level. (a) Federal excise taxes are imposed at... 29 Labor 3 2010-07-01 2010-07-01 false Excise taxes at the retail level. 779.262 Section 779.262...

Feasibility study of transanal total mesorectal excision

NARCIS (Netherlands)

Velthuis, S.; Boezem, P.B. van den; Peet, D.L. van der; Cuesta, M.A.; Sietses, C.

2013-01-01

BACKGROUND: Laparoscopic resection of colorectal cancers is a safe alternative to open surgery. The conversion rate to open surgery remains fairly constant but is associated with increased morbidity. A new approach to the surgical excision of rectal cancer is transanal total mesorectal excision

Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

Energy Technology Data Exchange (ETDEWEB)

Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon; Dulberger, Charles L.; Manlick, Angela J.; Keck, James L.; Cox, Michael M. (UW)

2012-03-16

The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

The role of DNA base excision repair in brain homeostasis and disease

DEFF Research Database (Denmark)

Akbari, Mansour; Morevati, Marya; Croteau, Deborah

2015-01-01

Chemical modification and spontaneous loss of nucleotide bases from DNA are estimated to occur at the rate of thousands per human cell per day. DNA base excision repair (BER) is a critical mechanism for repairing such lesions in nuclear and mitochondrial DNA. Defective expression or function of p...... energy homeostasis, mitochondrial function and cellular bioenergetics, with especially strong influence on neurological function. Further studies in this area could lead to novel approaches to prevent and treat human neurodegenerative disease....

Lower Lip Reconstruction after Wide Excision of a Malignancy with Barrel-Shaped Excision or the Webster Modification of the Bernard Operation

Directory of Open Access Journals (Sweden)

Hyung Joon Seo

2013-01-01

Full Text Available BackgroundBecause there are numerous methods for reconstruction of the lower lip, it is not easy to choose the optimal method. In choosing the surgical method for lower lip reconstruction, we obtained acceptable outcomes based on our treatment strategy, which included either a barrel-shaped excision or the Webster modification of the Bernard operation. We report on the surgical outcomes based on our treatment strategy.MethodsThis study included 26 patients who underwent lower lip reconstructive surgery from September 1996 to September 2010. The operation was done using either a barrel-shaped excision or the Webster modification, considering the location of the defect, the size of the defect, and the amount of residual tissue on the lateral side of the vermilion after excision.ResultsIn our series, 3 patients underwent a single barrel-shaped excision, and nine patients underwent a double barrel-shaped excision. In addition, the unilateral Webster modification was performed on in 6 patients, and there were eight cases of bilateral Webster modification. All of the patients except one were satisfied with the postoperative shape of the lip. In one case both recurrence and dehiscence occurred. One patient had a good postoperative lip shape, but had difficulty wearing a denture, and also underwent commissuroplasty. Furthermore, there were two patients who complained of drooling, and 4 with paresthesia.ConclusionsA soft tissue defect resulting from wide excision of a lower lip malignancy can be successfully reconstructed using only one of two surgical methods: the barrel-shaped excision or the Webster modification of the Bernard operation.

A metal-free DNA nuclease based on a cyclic peptide scaffold

Czech Academy of Sciences Publication Activity Database

Alkhader, S.; Ezra, A.; Kašpárková, Jana; Brabec, Viktor; Yavin, E.

2010-01-01

Roč. 21, č. 8 (2010), s. 1425-1431 ISSN 1043-1802 R&D Projects: GA AV ČR(CZ) IAA400040803; GA MŠk(CZ) LC06030; GA MŠk(CZ) ME08017; GA MŠk(CZ) OC08003; GA AV ČR(CZ) KAN200200651 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : DNA * cleavage * chemical nuclease Subject RIV: BO - Biophysics Impact factor: 5.002, year: 2010

Leishmania infantum EndoG is an endo/exo-nuclease essential for parasite survival.

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Eva Rico

Full Text Available EndoG, a member of the DNA/RNA non-specific ββα-metal family of nucleases, has been demonstrated to be present in many organisms, including Trypanosomatids. This nuclease participates in the apoptotic program in these parasites by migrating from the mitochondrion to the nucleus, where it takes part in the degradation of genomic DNA that characterizes this process. We now demonstrate that Leishmania infantum EndoG (LiEndoG is an endo-exonuclease that has a preferential 5' exonuclease activity on linear DNA. Regardless of its role during apoptotic cell death, this enzyme seems to be necessary during normal development of the parasites as indicated by the reduced growth rates observed in LiEndoG hemi-knockouts and their poor infectivity in differentiated THP-1 cells. The pro-life role of this protein is also corroborated by the higher survival rates of parasites that over-express this protein after treatment with the LiEndoG inhibitor Lei49. Taken together, our results demonstrate that this enzyme plays essential roles in both survival and death of Leishmania parasites.

Genotypic and antimicrobial characterisation of Propionibacterium acnes isolates from surgically excised lumbar disc herniations

DEFF Research Database (Denmark)

Rollason, Jess; McDowell, Andrew; Albert, Hanne B

2013-01-01

The anaerobic skin commensal Propionibacterium acnes is an underestimated cause of human infections and clinical conditions. Previous studies have suggested a role for the bacterium in lumbar disc herniation and infection. To further investigate this, five biopsy samples were surgically excised...... from each of 64 patients with lumbar disc herniation. P. acnes and other bacteria were detected by anaerobic culture, followed by biochemical and PCR-based identification. In total, 24/64 (38%) patients had evidence of P. acnes in their excised herniated disc tissue. Using recA and mAb typing methods...... isolate collection (63%) suggests that the role of P. acnes in lumbar disc herniation should not be readily dismissed....

Circumareolar Incision‑subdermal Tunneling Dissection for Excision ...

African Journals Online (AJOL)

Excision of multiple fibroadenomas (MF) in separate breast quadrants presents difficulties of number and location of incision(s) and extent of tissue dissection and may be associated with more complications and poorer cosmetic outcome. This is a report of excision of MF in multiple quadrants of the breast using a ...

Lumbar disc excision through fenestration

Directory of Open Access Journals (Sweden)

Sangwan S

2006-01-01

Full Text Available Background : Lumbar disc herniation often causes sciatica. Many different techniques have been advocated with the aim of least possible damage to other structures while dealing with prolapsed disc surgically in the properly selected and indicated cases. Methods : Twenty six patients with clinical symptoms and signs of prolapsed lumbar intervertebral disc having radiological correlation by MRI study were subjected to disc excision by interlaminar fenestration method. Results : The assessment at follow-up showed excellent results in 17 patients, good in 6 patients, fair in 2 patients and poor in 1 patient. The mean preoperative and postoperative Visual Analogue Scores were 9.34 ±0.84 and 2.19 ±0.84 on scale of 0-10 respectively. These were statistically significant (p value< 0.001, paired t test. No significant complications were recorded. Conclusion : Procedures of interlaminar fenestration and open disc excision under direct vision offers sufficient adequate exposure for lumbar disc excision with a smaller incision, lesser morbidity, shorter convalescence, early return to work and comparable overall results in the centers where recent laser and endoscopy facilities are not available.

DNA methylation in human fibroblasts following DNA damage and repair

International Nuclear Information System (INIS)

Kastan, M.B.

1984-01-01

Methylation of deoxycytidine (dCyd) incorporated by DNA excision repair synthesis in human diploid fibroblasts following damage with ultraviolet radiation (UV), N-methyl-N-nitrosourea, or N-acetoxy-2-acetylaminofluorene was studied utilizing [6- 3 H]dCyd to label repaired DNA specifically and high performance liquid chromatographic analysis to quantify the percentage of deoxycytidine converted to 5-methyldeoxycytidine (m 5 dCyd). In confluent, nondividing cells, methylation in repair patches induced by all three agents is slow and incomplete. Whereas after DNA replication a level of 3.4% m 5 dCyd is reached in less than 2 hours, following UV-stimulated repair synthesis in confluent cells it takes about 3 days to reach a level of approx.2.0% m 5 dCyd in the repair patch. This undermethylation of repair patches occurs throughout the genome. In cells from cultures in logarithmic-phase growth, m 5 dCyd formation in UV-induced repair patches occurs faster and to a greater extent, reaching a level of approx.2.7% in 10-20 hours. Pre-existing hypomethylated repair patches in confluent cells are methylated further when the cells are stimulated to divide; however, the repair patch may still not be fully methylated before cell division occurs. Thus DNA damage and repair may lead to heritable loss of methylation at some sites. The distribution within chromatin of m 5 dCyd in repair patches was also investigated. Over a wide range of extents of digestion by staphylococcal nuclease or deoxyribonuclease I, the level of hypomethylation in repaired DNA in nuclease sensitive and resistant regions of chromatin was constant relative to the genomic level of methylation in these regions. Similar conclusions were reached in experiments with isolated mononucleosomes

HETEROGENEITY OF POLYCLONAL IMMUNOGLOBULINS NUCLEASE ACTIVITY IN RHEUMATOID AND REACTIVE ARTHRITIS

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M. V. Volkova

2017-01-01

Full Text Available Catalytic properties of immunoglobulins are widely studied within recent years. It was found that nuclease activity of immunoglobulins is increased in systemic autoimmune diseases. Given some pathogenetic features of rheumatoid arthritis and reactive arthritis, it is appropriate to clarify the nature of nuclease activity in these diseases. Determination of DNAse activity of immunoglobulins with different DNA substrates, and search for specific substrates for distinct clinical entities could serve these purposes. The aim of present work is to determine DNase activity of the polyclonal class G immunoglobulins in rheumatoid and reactive arthritis using various methods.Different methods are used to evaluate nuclease activity. In this paper we present newly developed and modified techniques for determination of DNAse activity of polyclonal IgGs. Particular attention was paid to the electrophoretic method of DNase activity assessment. Polyclonal IgG isolated from blood serum of patients with rheumatoid arthritis and reactive arthritis were used for assays. In this study, we demonstrated the presence of an inhomogeneous DNase activity of immunoglobulins in relation to different substrates.Along with calf thymus DNA, we used bacterial plasmid DNA and PCR products based on bacterial gene sequences. Levels of DNase activity by rivanol clot method with calf thymus DNA as substrate proved to be higher in patients with rheumatoid arthritis than the control values (p < 0.01. DNase abzyme activity in patients with rheumatoid arthritis was elevated, as compared to the patients with reactive arthritis (p < 0.01.When examining ability of the IgG to hydrolyze procaryotic DNA (bacterial plasmid DNA and PCR products, based on bacterial genes, we obtained heterogeneous results. Different Ig samples showed varying degrees of DNA hydrolysis. Abzyme hydrolysis of DNA substrates longer than 700 bp was more pronounced, as compared to short DNA substrates (100 base pairs

Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases.

Directory of Open Access Journals (Sweden)

Anna Osiak

Full Text Available Gene knockout in murine embryonic stem cells (ESCs has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs. Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.

Dependence of u.v.-induced DNA excision repair on deoxyribonucleoside triphosphate concentrations in permeable human fibroblasts: a model for the inhibition of repair by hydroxyurea

International Nuclear Information System (INIS)

Hunting, D.J.; Dresler, S.L.

1985-01-01

We have tested the hypothesis that the inhibition by hydroxyurea of repair patch ligation and chromatin rearrangement during u.v.-induced DNA excision repair results from a reduction in cellular deoxyribonucleotide concentrations and not from a direct effect of hydroxyurea on the repair process. Using permeable human fibroblasts, we have shown that hydroxyurea has no direct effect on either repair synthesis or repair patch ligation. We also have shown that by reducing the deoxyribonucleoside triphosphate concentrations in the permeable cell reaction mixture, we can mimic the inhibition of repair patch ligation and chromatin rearrangement seen when u.v.-damaged intact confluent fibroblasts are treated with hydroxyurea. Our results are consistent with the concept that hydroxyurea inhibits DNA repair in intact cells by inhibiting deoxyribonucleotide synthesis through its effect on ribonucleotide reductase and, conversely, that continued deoxyribonucleotide synthesis is required for the excision repair of u.v.-induced DNA damage even in resting cells

Laparoscopic excision of a newborn rectal duplication cyst.

Science.gov (United States)

Hartin, Charles W; Lau, Stanley T; Escobar, Mauricio A; Glick, Philip L

2008-08-01

Congenital rectal duplication cyst is a rare entity treated with surgical excision. Without treatment, a rectal duplication cyst may cause a variety of complications, most notably, transforming into a malignancy. We report on a 7-week-old girl who was found to have a rectal duplication cyst. The rectal duplication cyst was successfully excised laparoscopically. Rectal duplication cysts are rare alimentary tract anomalies generally discovered during childhood. Complications include symptoms arising from the cyst and the possibility of malignant degeneration. They are typically managed by surgical excision.

Mitochondrial base excision repair assays

DEFF Research Database (Denmark)

Maynard, Scott; de Souza-Pinto, Nadja C; Scheibye-Knudsen, Morten

2010-01-01

The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur....... Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA...... glycosylases, AP endonuclease, DNA polymerase (POLgamma in mitochondria) and DNA ligase. This article outlines procedures for measuring oxidative damage formation and BER in mitochondria, including isolation of mitochondria from tissues and cells, protocols for measuring BER enzyme activities, gene...

Re-excision of margins before breast radiation-diagnostic or therapeutic?

International Nuclear Information System (INIS)

Chism, Derek B.; Freedman, Gary M.; Li, Tianyu; Anderson, Penny R.

2006-01-01

Purpose: To identify factors in breast cancer patients that predict the pathologic results of re-excision for close or positive margins and to determine the effect on local control. Methods and Materials: We divided 1,044 patients with Stage I-II breast cancer with a close (≤2 mm) or positive margin after initial excision into three groups. Group 1 included 199 patients without additional excision, Group 2 included 546 patients with re-excision found to be free of cancer, and Group 3 included 299 patients with re-excision and residual cancer. All patients were treated with radiotherapy with a median follow-up of 6.7 years. Results: The 10-year local control rate was 95% for Group 1 and 94% for Groups 2 and 3 (p = 0.788). Of the 846 patients, 65% had no residual disease on re-excision and 35% did have residual tumor. The factors significantly associated with positive re-excision findings were initial positive margins, positive nodes, Stage T2 tumor, and an extensive intraductal component. The 10-year local control rate was 95% for Group 2 vs. 91% for Group 3 (p = 0.038). Conclusion: The low recurrence rates seen in this study suggest that selected patients with non-negative margins, particularly those with a low risk of having residual disease at re-excision, may be treated with radiotherapy

Economic Impact of Imposing Excise Tax on Plastic Bottles of Drinks

Directory of Open Access Journals (Sweden)

Eugenia Mardanugraha

2018-03-01

Full Text Available This research simulates the effect of imposing excise tax on plastic container of drinks towards economic performance of beverage industry in Indonesia and governmentâ˘A ´Zs tax revenue. The results showed that by imposing excise tax on plastic cups and plastic bottles the government would lose tax revenue from value added tax (PPN and corporate income tax (PPh badan more than they gain additional revenue from excise tax. Hence, imposing excise tax on drink containers should serve a clear purpose and an undeniable reason. This paper recommends the government to develop proper excise infrastructure to extend the goods or services to be taxed. This paper also recommends the required stages for extending the excise tax.

Genome Editing with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline.

Science.gov (United States)

Sovová, Tereza; Kerins, Gerard; Demnerová, Kateřina; Ovesná, Jaroslava

2017-01-01

After induced mutagenesis and transgenesis, genome editing is the next step in the development of breeding techniques. Genome editing using site-directed nucleases - including meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system - is based on the mechanism of double strand breaks. The nuclease is directed to cleave the DNA at a specific place of the genome which is then repaired by natural repair mechanisms. Changes are introduced during the repair that are either accidental or can be targeted if a DNA template with the desirable sequence is provided. These techniques allow making virtually any change to the genome including specific DNA sequence changes, gene insertion, replacements or deletions with unprecedented precision and specificity while being less laborious and more straightforward compared to traditional breeding techniques or transgenesis. Therefore, the research in this field is developing quickly and, apart from model species, multiple studies have focused on economically important species and agronomically important traits that were the key subjects of this review. In plants, studies have been undertaken on disease resistance, herbicide tolerance, nutrient metabolism and nutritional value. In animals, the studies have mainly focused on disease resistance, meat production and allergenicity of milk. However, none of the promising studies has led to commercialization despite several patent applications. The uncertain legal status of genome-editing methods is one of the reasons for poor commercial development, as it is not clear whether the products would fall under the GMO regulation. We believe this issue should be clarified soon in order to allow promising methods to reach their full potential.

Union Women, the Tobacco Industry, and Excise Taxes

Science.gov (United States)

Balbach, Edith D.; Campbell, Richard B.

2009-01-01

Between 1987 and 1997, the tobacco industry used the issue of cigarette excise tax increases to create a political partnership with the Coalition of Labor Union Women (CLUW), a group representing female trade unionists in the U.S. This paper documents how the industry created this relationship and the lessons tobacco-control advocates can learn from the industry’s example, in order to mitigate possible unintended consequences of advocating excise tax increases In 1998, under the terms of the Master Settlement Agreement, the tobacco industry began making documents produced in litigation available publicly. Currently, approximately 50 million pages are available online, including substantial documentation of the industry–CLUW relationship. For this study, a comprehensive search of these documents was conducted. The tobacco industry encouraged CLUW’s opposition to excise tax increases by emphasizing the economic regressivity of these taxes, discussing excise taxes generically to deflect attention from cigarettes, and encouraging opposition to earmarking cigarette taxes to pay for specific programs. In addition, CLUW received at least $221,500 in financial support between 1987 and 1997 and in-kind support for its conferences, membership materials, and other services. Excise tax increases, if pursued without considering the impacts they may have on low-SES populations, may have unintended consequences. In this case, such proposals may have helped to create a relationship between CLUW and the tobacco industry. Because excise taxes are endorsed in the Framework Convention on Tobacco Control, tobacco-control advocates must understand how to build relationships with low-SES populations and mitigate potential alliances with the tobacco industry. PMID:19591750

Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis

DEFF Research Database (Denmark)

Lonowski, Lindsey A; Narimatsu, Yoshiki; Riaz, Anjum

2017-01-01

, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates...

Mung bean nuclease treatment increases capture specificity of microdroplet-PCR based targeted DNA enrichment.

Directory of Open Access Journals (Sweden)

Zhenming Yu

Full Text Available Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.

Black-hole excision with multiple grid patches

Energy Technology Data Exchange (ETDEWEB)

Thornburg, Jonathan [Max-Planck-Institut fuer Gravitationsphysik, Albert-Einstein-Institut, Am Muehlenberg 1, D-14476 Golm (Germany)

2004-08-07

When using black-hole excision to numerically evolve a black-hole spacetime with no continuous symmetries, most 3 + 1 finite differencing codes use a Cartesian grid. It is difficult to do excision on such a grid because the natural r = constant excision surface must be approximated either by a very different shape such as a contained cube, or by an irregular and non-smooth 'LEGO{sup 1} sphere' which may introduce numerical instabilities into the evolution. In this paper I describe an alternate scheme which uses multiple {l_brace}r x (angular coordinates){r_brace} grid patches, each patch using a different (nonsingular) choice of angular coordinates. This allows excision on a smooth r = constant 2-sphere. I discuss the key design choices in such a multiple-patch scheme, including the choice of ghost-zone versus internal-boundary treatment of the interpatch boundaries (I use a ghost-zone scheme), the number and shape of the patches (I use a 6-patch 'inflated-cube' scheme), the details of how the ghost zones are 'synchronized' by interpolation from neighbouring patches, the tensor basis for the Einstein equations in each patch, and the handling of non-tensor field variables such as the BSSN {gamma}-tilde{sup i} (I use a scheme which requires ghost zones which are twice as wide for the BSSN conformal factor {phi} as for {gamma}-tilde{sup i} and the other BSSN field variables). I present sample numerical results from a prototype implementation of this scheme. This code simulates the time evolution of the (asymptotically flat) spacetime around a single (excised) black hole, using fourth-order finite differencing in space and time. Using Kerr initial data with J/m{sup 2} = 0.6, I present evolutions to t {approx}> 1500m. The lifetime of these evolutions appears to be limited only by outer boundary instabilities, not by any excision instabilities or by any problems inherent to the multiple-patch scheme.

Black-hole excision with multiple grid patches

International Nuclear Information System (INIS)

Thornburg, Jonathan

2004-01-01

When using black-hole excision to numerically evolve a black-hole spacetime with no continuous symmetries, most 3 + 1 finite differencing codes use a Cartesian grid. It is difficult to do excision on such a grid because the natural r = constant excision surface must be approximated either by a very different shape such as a contained cube, or by an irregular and non-smooth 'LEGO 1 sphere' which may introduce numerical instabilities into the evolution. In this paper I describe an alternate scheme which uses multiple {r x (angular coordinates)} grid patches, each patch using a different (nonsingular) choice of angular coordinates. This allows excision on a smooth r = constant 2-sphere. I discuss the key design choices in such a multiple-patch scheme, including the choice of ghost-zone versus internal-boundary treatment of the interpatch boundaries (I use a ghost-zone scheme), the number and shape of the patches (I use a 6-patch 'inflated-cube' scheme), the details of how the ghost zones are 'synchronized' by interpolation from neighbouring patches, the tensor basis for the Einstein equations in each patch, and the handling of non-tensor field variables such as the BSSN Γ-tilde i (I use a scheme which requires ghost zones which are twice as wide for the BSSN conformal factor φ as for Γ-tilde i and the other BSSN field variables). I present sample numerical results from a prototype implementation of this scheme. This code simulates the time evolution of the (asymptotically flat) spacetime around a single (excised) black hole, using fourth-order finite differencing in space and time. Using Kerr initial data with J/m 2 = 0.6, I present evolutions to t ∼> 1500m. The lifetime of these evolutions appears to be limited only by outer boundary instabilities, not by any excision instabilities or by any problems inherent to the multiple-patch scheme

Effects of an extract from the sea squirt Ecteinascidia turbinata on DNA synthesis and excision repair in human fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Dunn, W.C.; Carrier, W.L.; Regan, J.D.

1982-01-01

An aqueous ethanol extract from the marine tunicate species Ecteinascidia turbinata was studied to determine its effect on semiconservative DNA synthesis in human skin fibroblast cultures as measured by (/sup 3/H) thymidine uptake in acid-insoluble cell fractions. In addition, the effect of this extract on DNA excision repair in ultraviolet light (254 nm) irradiated fibroblasts was measured by the bromodeoxyuridine photolysis assay, thymine dimer chromatography, and DNA single-strand break analysis on alkaline sucrose gradients. Repair inhibition was accompanied by an accumulation of single-strand DNA breaks which was enhanced by the addtion of 2 mM hydroxyurea. These results are discussed with respect to a mechanism of action of the marine tunicate extract at the level of DNA polymerases and are contrasted with previously studied inhibitory mechanisms of arabinofuranosyl nucleosides.

The Strategy of Excise Taxation of Tobacco Products in Ukraine

Directory of Open Access Journals (Sweden)

Pasichnyi Mykola D.

2017-06-01

Full Text Available The article is aimed at disclosing and improving approaches to the development of a strategy of excise tax policy in Ukraine, taking into account the foreign experience of harmonizing tax legislation in this sphere. An analysis of the implementation of the EU directives on the regulation of the minimum excise tax liability for the payment of excise taxes on tobacco products in the countries with transformational economies has been carried out. It has been found that, in cases of excessive tax pressure, the equilibrium of the market is disrupted, its shadow component is growing, and the overall economic efficiency level decreases. It has been determined that for the period of 2007-2016 the availability index for cigarettes in Ukraine decreased 2,3 times, which in some way demotivated their consumption. However, the change in the approach of calculation of ad valorem rate for the excise tax and introduction of the excise tax on the sub-excise goods sold by the retailers led to manipulative actions by the major actors in the market concerning the price of cigarettes, which impacted both the increase in the availability of cigarettes in 2016 and the decline in budget revenues. Regulation of the minimum excise duty is the most effective instrument of fiscal policy to achieve goals in the area of limitation of smoking.

Excision and crosslink repair of DNA and sister chromatid exchanges in cultured human fibroblasts with different repair capacities

Energy Technology Data Exchange (ETDEWEB)

Fujiwara, Y; Kano, Y; Paul, P; Goto, K; Yamamoto, K [Kobe Univ. (Japan). School of Medicine

1981-01-01

Xeroderma pigmentosum (XP) groups A to G lacked the initial stage of ultraviolet (UV) excision repair in the order of A = G > C > D > E asymptotically equals F, while the XP variant was weakly defective in the later repair steps. Killing sensitivities were in the orders of A >= G > D > C > E asymptotically equals F asymptotically equals variant > normal to UV, A = G > D > F > C = E > variant > normal to 4-nitroquinoline-1-oxide (4NQO), and A > C > D = E = F = variant > G = normal to decarbamoyl mitomycin-C(DCMC). The induced sister chromatid exchange (SCE) frequency was unrelated to the extent of repair deficiency. The SCE induction rate was consistently 3 - 6 fold higher by these UV-like mutagens in XP group A cells than in normal cells. However, repair-proficient Cockayne's syndrome (CS) cells showed a higher SCE induction by UV, which was normalized by NAD/sup +/, suggesting that chromatin lesions as well as DNA damage contribute to SCE. Two-step crosslink repair involves a first rapid half-excision and a second slow nucleotide-excision repair. Fanconi's anemia (FA) cells had an impaired first half-excision and were supersensitive to MC, but not to UV and DCMC. The SCE frequency induced by MC (1 hr) was higher in FA cells than in normal cells despite their normal response to DCMC, and vice versa in XP cells. FA cells lacked the first rapid decline and showed higher remaining SCEs. Thus, part of the crosslink seems to lead to SCE formation. Caffeine synergistically elevated UV-induced SCEs, but not UV induced mutations in V79 cells, implying that SCE may not necessarily involve mutation.

Excision and crosslink repair of DNA and sister chromatid exchanges in cultured human fibroblasts with different repair capacities

International Nuclear Information System (INIS)

Fujiwara, Yoshisada; Kano, Yoshio; Paul, P.; Goto, Kaoru; Yamamoto, Kazuo

1981-01-01

Xeroderma pigmentosum (XP) groups A to G lacked the initial stage of ultraviolet (UV) excision repair in the order of A = G > C > D > E asymptotically equals F, while the XP variant was weakly defective in the later repair steps. Killing sensitivities were in the orders of A >= G > D > C > E asymptotically equals F asymptotically equals variant > normal to UV, A = G > D > F > C = E > variant > normal to 4-nitroquinoline-1-oxide (4NQO), and A > C > D = E = F = variant > G = normal to decarbamoyl mitomycin-C(DCMC). The induced sister chromatid exchange (SCE) frequency was unrelated to the extent of repair deficiency. The SCE induction rate was consistently 3 - 6 fold higher by these UV-like mutagens in XP group A cells than in normal cells. However, repair-proficient Cockayne's syndrome (CS) cells showed a higher SCE induction by UV, which was normalized by NAD + , suggesting that chromatin lesions as well as DNA damage contribute to SCE. Two-step crosslink repair involves a first rapid half-excision and a second slow nucleotide-excision repair. Fanconi's anemia (FA) cells had an impaired first half-excision and were supersensitive to MC, but not to UV and DCMC. The SCE frequency induced by MC (1 hr) was higher in FA cells than in normal cells despite their normal response to DCMC, and vice versa in XP cells. FA cells lacked the first rapid decline and showed higher remaining SCEs. Thus, part of the crosslink seems to lead to SCE formation. Caffeine synergistically elevated UV-induced SCEs, but not UV induced mutations in V79 cells, implying that SCE may not necessarily involve mutation. (J.P.N.)

Excision and crosslink repair of DNA and sister chromatid exchanges in cultured human fibroblasts with different repair capacities

Energy Technology Data Exchange (ETDEWEB)

Fujiwara, Y.; Kano, Y.; Paul, P.; Goto, K.; Yamamoto, K. (Kobe Univ. (Japan). School of Medicine)

1981-01-01

Xeroderma pigmentosum (XP) groups A to G lacked the initial stage of ultraviolet (UV) excision repair in the order of A = G > C > D > E asymptotically equals F, while the XP variant was weakly defective in the later repair steps. Killing sensitivities were in the orders of A >= G > D > C > E asymptotically equals F asymptotically equals variant > normal to UV, A = G > D > F > C = E > variant > normal to 4-nitroquinoline-1-oxide (4NQO), and A > C > D = E = F = variant > G = normal to decarbamoyl mitomycin-C(DCMC). The induced sister chromatid exchange (SCE) frequency was unrelated to the extent of repair deficiency. The SCE induction rate was consistently 3 - 6 fold higher by these UV-like mutagens in XP group A cells than in normal cells. However, repair-proficient Cockayne's syndrome (CS) cells showed a higher SCE induction by UV, which was normalized by NAD/sup +/, suggesting that chromatin lesions as well as DNA damage contribute to SCE. Two-step crosslink repair involves a first rapid half-excision and a second slow nucleotide-excision repair. Fanconi's anemia (FA) cells had an impaired first half-excision and were supersensitive to MC, but not to UV and DCMC. The SCE frequency induced by MC (1 hr) was higher in FA cells than in normal cells despite their normal response to DCMC, and vice versa in XP cells. FA cells lacked the first rapid decline and showed higher remaining SCEs. Thus, part of the crosslink seems to lead to SCE formation. Caffeine synergistically elevated UV-induced SCEs, but not UV induced mutations in V79 cells, implying that SCE may not necessarily involve mutation.

Modulation of DNA polymerase beta-dependent base excision repair in cultured human cells after low dose exposure to arsenite

International Nuclear Information System (INIS)

Sykora, Peter; Snow, Elizabeth T.

2008-01-01

Base excision repair (BER) is crucial for development and for the repair of endogenous DNA damage. However, unlike nucleotide excision repair, the regulation of BER is not well understood. Arsenic, a well-established human carcinogen, is known to produce oxidative DNA damage, which is repaired primarily by BER, whilst high doses of arsenic can also inhibit DNA repair. However, the mechanism of repair inhibition by arsenic and the steps inhibited are not well defined. To address this question we have investigated the regulation of DNA polymerase β (Pol β) and AP endonuclease (APE1), in response to low, physiologically relevant doses of arsenic. GM847 lung fibroblasts and HaCaT keratinocytes were exposed to sodium arsenite, As(III), and mRNA, protein levels and BER activity were assessed. Both Pol β and APE1 mRNA exhibited significant dose-dependant down regulation at doses of As(III) above 1 μM. However, at lower doses Pol β mRNA and protein levels, and consequently, BER activity were significantly increased. In contrast, APE1 protein levels were only marginally increased by low doses of As(III) and there was no correlation between APE1 and overall BER activity. Enzyme supplementation of nuclear extracts confirmed that Pol β was rate limiting. These changes in BER correlated with overall protection against sunlight UV-induced toxicity at low doses of As(III) and produced synergistic toxicity at high doses. The results provide evidence that changes in BER due to low doses of arsenic could contribute to a non-linear, threshold dose response for arsenic carcinogenesis

RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures.

Science.gov (United States)

Theriot, Corey A; Hegde, Muralidhar L; Hazra, Tapas K; Mitra, Sankar

2010-06-04

The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (K(d) approximately 20 nM) via the common interacting interface (residues 312-349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CDelta78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CDelta78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome. Copyright 2010 Elsevier B.V. All rights reserved.

Posterior Endoscopic Excision of Os Trigonum in Professional National Ballet Dancers.

Science.gov (United States)

Ballal, Moez S; Roche, Andy; Brodrick, Anna; Williams, R Lloyd; Calder, James D F

2016-01-01

Previous studies have compared the outcomes after open and endoscopic excision of an os trigonum in patients of mixed professions. No studies have compared the differences in outcomes between the 2 procedures in elite ballet dancers. From October 2005 to February 2010, 35 professional ballet dancers underwent excision of a symptomatic os trigonum of the ankle after a failed period of nonoperative treatment. Of the 35 patients, 13 (37.1%) underwent endoscopic excision and 22 (62.9%) open excision. We compared the outcomes, complications, and time to return to dancing. The open excision group experienced a significantly greater incidence of flexor hallucis longus tendon decompression compared with the endoscopic group. The endoscopic release group returned to full dance earlier at a mean of 9.8 (range 6.5 to 16.1) weeks and those undergoing open excision returned to full dance at a mean of 14.9 (range 9 to 20) weeks (p = .001). No major complications developed in either group, such as deep infection or nerve or vessel injury. We have concluded that both techniques are safe and effective in the treatment of symptomatic os trigonum in professional ballet dancers. Endoscopic excision of the os trigonum offers a more rapid return to full dance compared with open excision. Copyright © 2016 American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.

CRISPR Genome Engineering for Human Pluripotent Stem Cell Research.

Science.gov (United States)

Chaterji, Somali; Ahn, Eun Hyun; Kim, Deok-Ho

2017-01-01

The emergence of targeted and efficient genome editing technologies, such as repurposed bacterial programmable nucleases (e.g., CRISPR-Cas systems), has abetted the development of cell engineering approaches. Lessons learned from the development of RNA-interference (RNA-i) therapies can spur the translation of genome editing, such as those enabling the translation of human pluripotent stem cell engineering. In this review, we discuss the opportunities and the challenges of repurposing bacterial nucleases for genome editing, while appreciating their roles, primarily at the epigenomic granularity. First, we discuss the evolution of high-precision, genome editing technologies, highlighting CRISPR-Cas9. They exist in the form of programmable nucleases, engineered with sequence-specific localizing domains, and with the ability to revolutionize human stem cell technologies through precision targeting with greater on-target activities. Next, we highlight the major challenges that need to be met prior to bench-to-bedside translation, often learning from the path-to-clinic of complementary technologies, such as RNA-i. Finally, we suggest potential bioinformatics developments and CRISPR delivery vehicles that can be deployed to circumvent some of the challenges confronting genome editing technologies en route to the clinic.

LEM-3 is a midbody-tethered DNA nuclease that resolves chromatin bridges during late mitosis.

Science.gov (United States)

Hong, Ye; Sonneville, Remi; Wang, Bin; Scheidt, Viktor; Meier, Bettina; Woglar, Alexander; Demetriou, Sarah; Labib, Karim; Jantsch, Verena; Gartner, Anton

2018-02-20

Faithful chromosome segregation and genome maintenance requires the removal of all DNA bridges that physically link chromosomes before cells divide. Using C. elegans embryos we show that the LEM-3/Ankle1 nuclease defines a previously undescribed genome integrity mechanism by processing DNA bridges right before cells divide. LEM-3 acts at the midbody, the structure where abscission occurs at the end of cytokinesis. LEM-3 localization depends on factors needed for midbody assembly, and LEM-3 accumulation is increased and prolonged when chromatin bridges are trapped at the cleavage plane. LEM-3 locally processes chromatin bridges that arise from incomplete DNA replication, unresolved recombination intermediates, or the perturbance of chromosome structure. Proper LEM-3 midbody localization and function is regulated by AIR-2/Aurora B kinase. Strikingly, LEM-3 acts cooperatively with the BRC-1/BRCA1 homologous recombination factor to promote genome integrity. These findings provide a molecular basis for the suspected role of the LEM-3 orthologue Ankle1 in human breast cancer.

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

Science.gov (United States)

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

2013-07-01

Customized DNA-binding domains made using transcription activator-like effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in various organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or downregulate expression of endogenous genes in human cells and plants. This unit describes a detailed protocol for the recently described fast ligation-based automatable solid-phase high-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multichannel pipet. Using the automated approach, a single researcher can construct up to 96 DNA fragments encoding TALE repeat arrays of various lengths in a single day, and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in a week or less. Plasmids required for FLASH are available by request from the Joung lab (http://eGenome.org). This unit also describes improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) web server (http://ZiFiT.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high throughput. © 2013 by John Wiley & Sons, Inc.

Excision repair in MUT-mutants of Proteus mirabilis after UV-irradiation

International Nuclear Information System (INIS)

Stoerl, K.; Mund, C.

1977-01-01

The behaviour of MUT-mutants of P.mirabilis to perform certain steps of excision repair after U.V.-irradiation is described. MUT-mutants introduce single-strand breaks in the DNA immediately after U.V.-irradiation, but their ability to excise pyrimidine dimers from the DNA is very diminished. Moreover, they are not able to accomplish the excision repair by rejoining of the single-strand breaks. The connection between the incomplete excision repair and the mutator phenotype of these mutants is discussed. (author)

Elliptical excisions: variations and the eccentric parallelogram.

Science.gov (United States)

Goldberg, Leonard H; Alam, Murad

2004-02-01

The elliptical (fusiform) excision is a basic tool of cutaneous surgery. To assess the design, functionality, ease of construction, and aesthetic outcomes of the ellipse. A systematic review of elliptical designs and their site-specific benefits and limitations. In particular, we consider the (1). context of prevailing relaxed skin tension lines and tissue laxity; and (2). removal of the smallest possible amount of tissue around the lesion and in the "dog-ears." Attention is focused on intuitive methods that can be reproducibly planned and executed. Elliptical variations are easily designed and can be adapted to many situations. The eccentric parallelogram excision is offered as a new technique that minimizes notching and focal tension in the center of an elliptical closure. Conclusion The elliptical (fusiform) excision is an efficient, elegant, and versatile technique that will remain a mainstay of the cutaneous surgical armamentarium.

TALE nucleases and next generation GM crops.

KAUST Repository

Mahfouz, Magdy M.

2011-04-01

Site-specific and adaptable DNA binding domains are essential modules to develop genome engineering technologies for crop improvement. Transcription activator-like effectors (TALEs) proteins are used to provide a highly specific and adaptable DNA binding modules. TALE chimeric nucleases (TALENs) were used to generate site-specific double strand breaks (DSBs) in vitro and in yeast, Caenorhabditis elegans, mammalian and plant cells. The genomic DSBs can be generated at predefined and user-selected loci and repaired by either the non-homologous end joining (NHEJ) or homology dependent repair (HDR). Thus, TALENs can be used to achieve site-specific gene addition, stacking, deletion or inactivation. TALE-based genome engineering tools should be powerful to develop new agricultural biotechnology approaches for crop improvement. Here, we discuss the recent research and the potential applications of TALENs to accelerate the generation of genomic variants through targeted mutagenesis and to produce a non-transgenic GM crops with the desired phenotype.

Higher Volume at Time of Breast Conserving Surgery Reduces Re-Excision in DCIS

Directory of Open Access Journals (Sweden)

J. H. Wolf

2011-01-01

Full Text Available Purpose. The purpose of this study was to compare the surgical and pathological variables which impact rate of re-excision following breast conserving therapy (BCS with or without concurrent additional margin excision (AM. Methods. The pathology database was queried for all patients with DCIS from January 2004 to September 2008. Pathologic assessment included volume of excision, subtype, size, distance from margin, grade, necrosis, multifocality, calcifications, and ER/PR status. Results. 405 cases were identified and 201 underwent BCS, 151-BCS-AM, and 53-mastectomy. Among the 201 BCS patients, 190 underwent re-excision for close or involved margins. 129 of these were treated with BCS and 61 with BCS-AM (P<.0001. The incidence of residual DCIS in the re-excision specimens was 32% (n=65 for BCS and 22% (n=33 for BCS-AM (P<.05. For both the BCS and the BCS-AM cohorts, volume of tissue excised is inversely correlated to the rate of re-excision (P=.0284. Multifocality (P=.0002 and ER status (P=.0382 were also significant predictors for rate of re-excision and variation in surgical technique was insignificant. Conclusions. The rate of positive margins, re-excision, and residual disease was significantly higher in patients with lower volume of excision. The performance of concurrent additional margin excision increases the efficacy of BCS for DCIS.

Identification of genes and proteins involved in excision repair of human cells

International Nuclear Information System (INIS)

Hoeijmakers, J.H.J.; Westerveld, A.; Van Duin, M.; Vermeulen, W.; Odijk, H.; De Wit, J.; Bootsma, D.

1986-01-01

The autosomal, recessive disorder xeroderma pigmentosum (XP) is characterized by extreme sensitivity of the skin to sun exposure and prediposition to skin cancer. The basic defect in most XP patients is thought to reside in an inefficient removal of UV-induced lesions in the DNA by excision repair. The biochemical complexity of this process is amply illustrated by the fact that so far nine complementary groups within this syndrome have been identified. Despite extensive research, none of these genes or proteins involved have been isolated. Using a microinjection assay system the authors identified components in crude cell extracts that transiently correct the defect in (injected) fibroblasts of all excision-deficient XP complementation groups, as indicated by temporary restoration of UV-induced unscheduled DNA synthesis. This correction is complementation group specific, since it is only found when extracts from complementing XP cells are injected. After incubation of extracts with proteinase K the XP-A and KP-G correcting activities were lost, indicating that the complementation is due to proteins. The XP-A correcting protein was found to precipitate between 30 and 60% ammonium sulfate saturation. Furthermore this protein binds to DEAE-cellulose and to (UV-irradiated) double-strand (ds) DNA attached to cellulose. The latter affinity chromatography step allows a considerable purification, since less than 1% of the proteins applied to such columns is retained. It has to be established whether the XP-A correcting proteins binds by itself or via other proteins to the UV-irradiated DNA and whether it also binds to nonirradiated (ds or ss) DNA. Similar experiments with the XP-G correcting protein are in progress

Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster.

Science.gov (United States)

Simoni, Alekos; Siniscalchi, Carla; Chan, Yuk-Sang; Huen, David S; Russell, Steven; Windbichler, Nikolai; Crisanti, Andrea

2014-06-01

Selfish genes are DNA elements that increase their rate of genetic transmission at the expense of other genes in the genome and can therefore quickly spread within a population. It has been suggested that selfish elements could be exploited to modify the genome of entire populations for medical and ecological applications. Here we report that transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) can be engineered into site-specific synthetic selfish elements (SSEs) and demonstrate their transmission of up to 70% in the Drosophila germline. We show here that SSEs can spread via DNA break-induced homologous recombination, a process known as 'homing' similar to that observed for homing endonuclease genes (HEGs), despite their fundamentally different modes of DNA binding and cleavage. We observed that TALEN and ZFN have a reduced capability of secondary homing compared to HEG as their repetitive structure had a negative effect on their genetic stability. The modular architecture of ZFNs and TALENs allows for the rapid design of novel SSEs against specific genomic sequences making them potentially suitable for the genetic engineering of wild-type populations of animals and plants, in applications such as gene replacement or population suppression of pest species. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Protective Effect of Diphlorethohydroxycarmalol against Ultraviolet B Radiation-Induced DNA Damage by Inducing the Nucleotide Excision Repair System in HaCaT Human Keratinocytes

Directory of Open Access Journals (Sweden)

Mei Jing Piao

2015-09-01

Full Text Available We investigated the protective properties of diphlorethohydroxycarmalol (DPHC, a phlorotannin, against ultraviolet B (UVB radiation-induced cyclobutane pyrimidine dimers (CPDs in HaCaT human keratinocytes. The nucleotide excision repair (NER system is the pathway by which cells identify and repair bulky, helix-distorting DNA lesions such as ultraviolet (UV radiation-induced CPDs and 6-4 photoproducts. CPDs levels were elevated in UVB-exposed cells; however, this increase was reduced by DPHC. Expression levels of xeroderma pigmentosum complementation group C (XPC and excision repair cross-complementing 1 (ERCC1, which are essential components of the NER pathway, were induced in DPHC-treated cells. Expression of XPC and ERCC1 were reduced following UVB exposure, whereas DPHC treatment partially restored the levels of both proteins. DPHC also increased expression of transcription factor specificity protein 1 (SP1 and sirtuin 1, an up-regulator of XPC, in UVB-exposed cells. DPHC restored binding of the SP1 to the XPC promoter, which is reduced in UVB-exposed cells. These results indicate that DPHC can protect cells against UVB-induced DNA damage by inducing the NER system.

First observation of excision and integration in Class 1 integron in ...

African Journals Online (AJOL)

So in this study, we tested in S. aureus, the class 1 integron mediated excision and integration. We first asked 8 plasmids from previous studies, then established some transformants and perform the excision and integration reaction. As the results revealed, we observed positive excision assay, which had been confirmed by ...

Computational Characterization of Small Molecules Binding to the Human XPF Active Site and Virtual Screening to Identify Potential New DNA Repair Inhibitors Targeting the ERCC1-XPF Endonuclease

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Francesco Gentile

2018-04-01

Full Text Available The DNA excision repair protein ERCC-1-DNA repair endonuclease XPF (ERCC1-XPF is a heterodimeric endonuclease essential for the nucleotide excision repair (NER DNA repair pathway. Although its activity is required to maintain genome integrity in healthy cells, ERCC1-XPF can counteract the effect of DNA-damaging therapies such as platinum-based chemotherapy in cancer cells. Therefore, a promising approach to enhance the effect of these therapies is to combine their use with small molecules, which can inhibit the repair mechanisms in cancer cells. Currently, there are no structures available for the catalytic site of the human ERCC1-XPF, which performs the metal-mediated cleavage of a DNA damaged strand at 5′. We adopted a homology modeling strategy to build a structural model of the human XPF nuclease domain which contained the active site and to extract dominant conformations of the domain using molecular dynamics simulations followed by clustering of the trajectory. We investigated the binding modes of known small molecule inhibitors targeting the active site to build a pharmacophore model. We then performed a virtual screening of the ZINC Is Not Commercial 15 (ZINC15 database to identify new ERCC1-XPF endonuclease inhibitors. Our work provides structural insights regarding the binding mode of small molecules targeting the ERCC1-XPF active site that can be used to rationally optimize such compounds. We also propose a set of new potential DNA repair inhibitors to be considered for combination cancer therapy strategies.

Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci.

Science.gov (United States)

Canver, Matthew C; Lessard, Samuel; Pinello, Luca; Wu, Yuxuan; Ilboudo, Yann; Stern, Emily N; Needleman, Austen J; Galactéros, Frédéric; Brugnara, Carlo; Kutlar, Abdullah; McKenzie, Colin; Reid, Marvin; Chen, Diane D; Das, Partha Pratim; A Cole, Mitchel; Zeng, Jing; Kurita, Ryo; Nakamura, Yukio; Yuan, Guo-Cheng; Lettre, Guillaume; Bauer, Daniel E; Orkin, Stuart H

2017-04-01

Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, which is associated with red-blood-cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false-positive regions, thus emphasizing the importance of off-target analysis in the design of saturating-mutagenesis experiments. Together, these data establish a widely applicable high-throughput and high-resolution methodology to identify minimal functional sequences within large disease- and trait-associated regions.

Technique for laparoscopic autonomic nerve preserving total mesorectal excision.

Science.gov (United States)

Breukink, S O; Pierie, J P E N; Hoff, C; Wiggers, T; Meijerink, W J H J

2006-05-01

With the introduction of total mesorectal excision (TME) for treatment of rectal cancer, the prognosis of patients with rectal cancer is improved. With this better prognosis, there is a growing awareness about the quality of life of patients after rectal carcinoma. Laparoscopic total mesorectal excision (LTME) for rectal cancer offers several advantages in comparison with open total mesorectal excision (OTME), including greater patient comfort and an earlier return to daily activities while preserving the oncologic radicality of the procedure. Moreover, laparoscopy allows good exposure of the pelvic cavity because of magnification and good illumination. The laparoscope seems to facilitate pelvic dissection including identification and preservation of critical structures such as the autonomic nervous system. The technique for laparoscopic autonomic nerve preserving total mesorectal excision is reported. A three- or four-port technique is used. Vascular ligation, sharp mesorectal dissection and identification and preservation of the autonomic pelvic nerves are described.

P element excision in drosophila melanogaster and related drosophilids

Science.gov (United States)

The frequency of P element excision and the structure of the resulting excision products were determined in three drosophilid species, Drosophila melanogaster, D. virilis, and Chymomyza procnemis. A transient P element mobility assay was conducted in the cells of developing insect embryos, but unlik...

Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

KAUST Repository

Sobhy, M.; Joudeh, L.; Huang, X.; Takahashi, Masateru; Hamdan, S.

2013-01-01

Human flap endonuclease 1 (FEN1), one of the structure-specific 5' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5' nuclease mechanisms. This is achieved by coordinating threading of the 5' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection ofDNA junctions from nonspecific bending and cleavage. 2013 The Authors.

Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

KAUST Repository

Sobhy, M.

2013-06-06

Human flap endonuclease 1 (FEN1), one of the structure-specific 5\\' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5\\' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5\\' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5\\' nuclease mechanisms. This is achieved by coordinating threading of the 5\\' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5\\' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection ofDNA junctions from nonspecific bending and cleavage. 2013 The Authors.

Excision of x-ray-induced thymine damage in chromatin from heated cells

International Nuclear Information System (INIS)

Warters, R.L.; Roti Roti, J.L.

1979-01-01

Experiments were performed to distinguish between two possible modes of hyperthermia-induced inhibition of thymine base damage excision from the DNA of CHO cells: (1) heat denaturation of excision enzyme(s) or (2) heat-induced alteration of the substrate for damage excision (chromatin). While hyperthermia (45 0 C, 15 min) had no apparent effect on the capacity of the excision enzymes to excise damage from DNA it had a dramatic effect (ca. 80% inhibition) on the ability of chromatin to serve as a substrate for unheated enzymes. These results suggest that hyperthermia-induced radiosensitization of CHO cells may be due primarily to lesions in the cellular chromatin

29 CFR 779.263 - Excise taxes not at the retail level.

Science.gov (United States)

2010-07-01

... ACT AS APPLIED TO RETAILERS OF GOODS OR SERVICES Employment to Which the Act May Apply; Enterprise Coverage Excise Taxes § 779.263 Excise taxes not at the retail level. There are also a wide variety of... 29 Labor 3 2010-07-01 2010-07-01 false Excise taxes not at the retail level. 779.263 Section 779...

Targeted gene therapy of xeroderma pigmentosum cells using meganuclease and TALEN™.

Directory of Open Access Journals (Sweden)

Aurélie Dupuy

Full Text Available Xeroderma pigmentosum group C (XP-C is a rare human syndrome characterized by hypersensitivity to UV light and a dramatic predisposition to skin neoplasms. XP-C cells are deficient in the nucleotide excision repair (NER pathway, a complex process involved in the recognition and removal of DNA lesions. Several XPC mutations have been described, including a founder mutation in North African patients involving the deletion of a TG dinucleotide (ΔTG located in the middle of exon 9. This deletion leads to the expression of an inactive truncated XPC protein, normally involved in the first step of NER. New approaches used for gene correction are based on the ability of engineered nucleases such as Meganucleases, Zinc-Finger nucleases or TALE nucleases to accurately generate a double strand break at a specific locus and promote correction by homologous recombination through the insertion of an exogenous DNA repair matrix. Here, we describe the targeted correction of the ΔTG mutation in XP-C cells using engineered meganuclease and TALEN™. The methylated status of the XPC locus, known to inhibit both of these nuclease activities, led us to adapt our experimental design to optimize their in vivo efficacies. We show that demethylating treatment as well as the use of TALEN™ insensitive to CpG methylation enable successful correction of the ΔTG mutation. Such genetic correction leads to re-expression of the full-length XPC protein and to the recovery of NER capacity, attested by UV-C resistance of the corrected cells. Overall, we demonstrate that nuclease-based targeted approaches offer reliable and efficient strategies for gene correction.

Transvaginal sling excision: tips and tricks.

Science.gov (United States)

Clifton, Marisa M; Goldman, Howard B

2017-01-01

Complications of synthetic midurethral sling surgery include bladder outlet obstruction, mesh extrusion, and vaginal pain. A treatment of these complications is transvaginal mesh removal. The objectives of this video are to present cases of complications after sling placement and describe techniques to help with successful sling removal. Three patients are presented in this video. One experienced urinary hesitancy and was found to have bladder outlet obstruction on urodynamic study. The second patient presented to the clinic with diminished force of stream and significant dyspareunia. The last patient presented with mesh extrusion. After discussion of management options, all three patients wished to pursue transvaginal sling excision. All patients had successful removal of a portion of their synthetic midurethral sling. This video presents techniques to aide with dissection, mesh excision and prevention of further mesh complications. These include using an individualized surgical technique based on patient presentation and surgeon expertise, planning surgical incisions based on where mesh can be identified or palpated, using a cystoscope sheath or urethral dilator to identify any bladder outlet obstruction, and using a knife blade to identify mesh from surrounding tissue. Sling excision can be successfully performed with careful surgical technique and dissection.

Ulnar nerve entrapment complicating radial head excision

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Kevin Parfait Bienvenu Bouhelo-Pam

Full Text Available Introduction: Several mechanisms are involved in ischemia or mechanical compression of ulnar nerve at the elbow. Presentation of case: We hereby present the case of a road accident victim, who received a radial head excision for an isolated fracture of the radial head and complicated by onset of cubital tunnel syndrome. This outcome could be the consequence of an iatrogenic valgus of the elbow due to excision of the radial head. Hitherto the surgical treatment of choice it is gradually been abandoned due to development of radial head implant arthroplasty. However, this management option is still being performed in some rural centers with low resources. Discussion: The radial head plays an important role in the stability of the elbow and his iatrogenic deformity can be complicated by cubital tunnel syndrome. Conclusion: An ulnar nerve release was performed with favorable outcome. Keywords: Cubital tunnel syndrome, Peripheral nerve palsy, Radial head excision, Elbow valgus

Similar distributions of repaired sites in chromatin of normal and xeroderma pigmentosum variant cells damaged by ultraviolet light

International Nuclear Information System (INIS)

Cleaver, J.E.

1979-01-01

Excision repair of damage from ultraviolet light in both normal and xeroderma pigmentosum variant fibroblasts at early times after irradiation occurred preferentially in regions of DNA accessible to micrococcal nuclease digestion. These regions are predominantly the linker regions between nucleosomes in chromatin. The alterations reported at polymerization and ligation steps of excision repair in the variant are therefore not associated with changes in the relative distributions of repair sites in linker and core particle regions of DNA. (Auth.)

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

KAUST Repository

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

2011-01-01

Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions

Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula

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Lomate, Purushottam R.; Bonning, Bryony C.

2016-01-01

Stink bugs negatively impact numerous plant species of agricultural and horticultural importance. While efforts to develop effective control measures are underway, the unique digestive physiology of these pests presents a significant hurdle for either protein- or nucleotide-based management options. Here we report the comparative biochemical and proteomic characterization of proteases and nucleases from the gut, salivary gland and saliva of the southern green stink bug, Nezara viridula. The pH optimum for protease activity was acidic (5 to 6) in the gut with the primary proteases being cysteine proteases, and alkaline (8 to 9) in the saliva and salivary gland with the primary proteases being serine proteases. The serine proteases in saliva differ biochemically from trypsin and chymotrypsin, and the cathepsins in the gut and saliva showed distinct properties in inhibitor assays. Nuclease activity (DNase, RNase, dsRNase) was concentrated in the salivary gland and saliva with negligible activity in the gut. The most abundant proteins of the gut (530) and salivary gland (631) identified by proteomic analysis included four gut proteases along with eight proteases and one nuclease from the salivary gland. Understanding of N. viridula digestive physiology will facilitate the design of new strategies for management of this significant pest. PMID:27282882

Is surgical excision necessary for the treatment of Granulomatous lobular mastitis?

Science.gov (United States)

Shin, Young Duck; Park, Sung Su; Song, Young Jin; Son, Seung-Myoung; Choi, Young Jin

2017-07-24

We aimed to investigate the role of surgical excision in treating granulomatous lobular mastitis. We performed a retrospective chart review of patients with granulomatous lobular mastitis treated from March 2008 to March 2014. We analyzed clinical features and therapeutic modalities and compared the patient outcomes based on treatment. During the study period, a total of 34 patients were diagnosed with granulomatous lobular mastitis and treated. Initial treatments included wide excision (18), oral steroids after incision and drainage (14), and antibiotic therapy (2). The patients receiving only antibiotic therapy showed no improvement after 1 month and wide excision was then performed. Wide excision resulted in nine case of delayed wound healing with fistula. These patients were treated with oral steroids for 1.5-5 months, with subsequent improvement. Overall, 11 out of 20 patients who had underwent wide excision showed improvement without additional treatment. Fourteen patients who had initially received oral steroids for 1 to 6 months (average, 2.8 months) after incision and drainage showed complete remission. During the median follow-up period with 45.5 months (range, 22-98 months), six patients (17.6%) experienced recurrence. Wide excision group experienced recurrence in five (25%) and steroid and drainage group experienced recurrence in one (7.1%). All six recurrences responded to additional steroid therapy for average 3.5 months. Most wide excision group left extensive breast scarring with deformation that was not in steroid and drainage group. Wide excision resulted high recurrence than steroid and drainage group and left extensive scarring. Steroid therapy with or without abscess drainage may be the first choice of treatment for majority cases with granulomatous lobular mastitis.

Robotic Extramucosal Excision of Bladder Wall Leiomyoma

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Khalid E. Al-Othman

2014-01-01

Full Text Available Introduction: Multiple case reports and reviews have been described in the literature for bladder wall leiomyoma resection via different approaches. The minimally invasive partial cystectomy remains the most widely accepted technique; however, case reports for enucleation of bladder wall leiomyoma have also been described. The purpose of this video is to demonstrate the robotic extramucosal excision of a bladder wall leiomyoma, without cystotomy, but with complete removal of the muscular layer. Materials and Methods: A 35-year old male present with lower urinary tract symptoms and imaging showed bladder wall mass with histopathology showed leiomyoma. The patient consented for mass excision with the possibility of a partial cystectomy. The patient was placed in the supine, 30-degree Trendelenburg position during the procedure. A total of 4 ports were inserted. A 3-arm da Vinci robotic surgical system was docked, and the arms were connected. Extramucosal excision was accomplished without cystotomy and muscle approximation was achieved by 2 0 Vicryle. Result: The operative time was 90 minutes, blood loss of approximately 50mL and the patient was discharged after 72 hours with no immediate complications and a 6 months follow-up showed no recurrence. Conclusion: Such a technique results in complete excision of the tumor, without cystotomy, and also maintains an intact mucosa. These steps, in addition to decreasing the risk of local recurrence, also shorten the period of postoperative catheterization and hospitalization.

Nucleotide Excision DNA Repair is Associated with Age-Related Vascular Dysfunction

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Durik, Matej; Kavousi, Maryam; van der Pluijm, Ingrid; Isaacs, Aaron; Cheng, Caroline; Verdonk, Koen; Loot, Annemarieke E.; Oeseburg, Hisko; Musterd-Bhaggoe, Usha; Leijten, Frank; van Veghel, Richard; de Vries, Rene; Rudez, Goran; Brandt, Renata; Ridwan, Yanto R.; van Deel, Elza D.; de Boer, Martine; Tempel, Dennie; Fleming, Ingrid; Mitchell, Gary F.; Verwoert, Germaine C.; Tarasov, Kirill V.; Uitterlinden, Andre G.; Hofman, Albert; Duckers, Henricus J.; van Duijn, Cornelia M.; Oostra, Ben A.; Witteman, Jacqueline C.M.; Duncker, Dirk J.; Danser, A.H. Jan; Hoeijmakers, Jan H.; Roks, Anton J.M.

2012-01-01

Background Vascular dysfunction in atherosclerosis and diabetes, as observed in the aging population of developed societies, is associated with vascular DNA damage and cell senescence. We hypothesized that cumulative DNA damage during aging contributes to vascular dysfunction. Methods and Results In mice with genomic instability due to the defective nucleotide excision repair genes ERCC1 and XPD (Ercc1d/− and XpdTTD mice), we explored age-dependent vascular function as compared to wild-type mice. Ercc1d/− mice showed increased vascular cell senescence, accelerated development of vasodilator dysfunction, increased vascular stiffness and elevated blood pressure at very young age. The vasodilator dysfunction was due to decreased endothelial eNOS levels as well as impaired smooth muscle cell function, which involved phosphodiesterase (PDE) activity. Similar to Ercc1d/− mice, age-related endothelium-dependent vasodilator dysfunction in XpdTTD animals was increased. To investigate the implications for human vascular disease, we explored associations between single nucleotide polymorphisms (SNPs) of selected nucleotide excision repair genes and arterial stiffness within the AortaGen Consortium, and found a significant association of a SNP (rs2029298) in the putative promoter region of DDB2 gene with carotid-femoral pulse wave velocity. Conclusions Mice with genomic instability recapitulate age-dependent vascular dysfunction as observed in animal models and in humans, but with an accelerated progression, as compared to wild type mice. In addition, we found associations between variations in human DNA repair genes and markers for vascular stiffness which is associated with aging. Our study supports the concept that genomic instability contributes importantly to the development of cardiovascular disease. PMID:22705887

Impact of cigarette taxation policy on excise revenues and cigarette consumption in Uzbekistan

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Konstantin S. Krasovsky

2013-05-01

Full Text Available BACKGROUND: In 2012, Uzbekistan ratified the Framework Convention on Tobacco Control, which states that price and tax measures are an effective means of reducing tobacco consumption. We aimed to explore the effect of taxation policies on revenues and cigarette consumption. METHODS: Data on tax rates, revenues, cigarette sales were taken from national reports. To forecast potential revenues, a scenario analysis was performed. RESULTS: In 1991-2004, ad valorem excise system was in place in Uzbekistan, which was later replaced by the specific excise system. In 1997-2011, the nominal average excise has increased by a factor of twenty, but in real terms, after a sharp increase in 1999, average excise declined annually and increased only in 2010-2011. Annual cigarette sales per capita of adult population in 1999-2007 constituted 17-25 cigarette packs, while in 2008-2011 it increased to 30-37 packs. Four scenarios of excise tax increases in 2012 were developed: one actual scenario based on the rates effective in Uzbekistan in 2012, and three hypothetical ones anticipating excise rates increase by 1.5, 2 and 3-fold. With actual excise increase in 2012, the inflation-adjusted budget revenues would grow by 5%, and with three hypothetical - by 17%, 35% and 66% respectively, despite the decline of tax-paid cigarette sales. CONCLUSION: Stabilization or reduction in cigarette excises in Uzbekistan in 2002-2008 led to a decline in real excise revenues and the growth of cigarette sales. In 1999 and 2010-2011, excises were significantly increased and the real revenues have risen, despite the decline in cigarette sales. As cigarette prices are low, the illegal outflow of cigarettes from Uzbekistan apparently exceeds the illegal inflow. A significant increase in cigarette excise (1.5-3 fold can both increase budget revenues and reduce cigarette consumption, with greater increase yielding more benefits.

Nuclear translocation contributes to regulation of DNA excision repair activities

DEFF Research Database (Denmark)

Knudsen, Nina Østergaard; Andersen, Sofie Dabros; Lützen, Anne

2009-01-01

for regulation of nuclear import that is necessary for proper localization of the repair proteins. This review summarizes the current knowledge on nuclear import mechanisms of DNA excision repair proteins and provides a model that categorizes the import by different mechanisms, including classical nuclear import......DNA mutations are circumvented by dedicated specialized excision repair systems, such as the base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways. Although the individual repair pathways have distinct roles in suppressing changes in the nuclear DNA......, it is evident that proteins from the different DNA repair pathways interact [Y. Wang, D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, J. Qin, BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures, Genes Dev. 14 (2000) 927-939; M. Christmann, M...

Development of excise on automotive fuels in the neighbouring countries

International Nuclear Information System (INIS)

Bleijenberg, A.N.; Janse, P.

1994-04-01

The political options for the Netherlands to implement the pricing policy for road traffic by means of an increase of excise, as formulated in the Second Transport Structure Plan (SVV-2), are limited by the excise level in neighbouring countries. Therefore, a study on the title subject has been carried out. First, an overview is given of the historical development of sales prices since 1980 for Belgium, Germany, Luxembourg and France with special attention for the large price differences. Next, insight into the effects of a price difference at the borders is given. Subsequently the development in the policy of the European Union with regard to harmonization of excise tariffs is discussed. Environmental organizations in Belgium and Luxembourg were contacted to learn about the expected development of excises in those countries. 5 figs., 6 tabs., 4 appendices, 10 refs

Crystallization and preliminary crystallographic analysis of an Escherichia coli-selected mutant of the nuclease domain of the metallonuclease colicin E7

International Nuclear Information System (INIS)

Czene, Anikó; Tóth, Eszter; Gyurcsik, Béla; Otten, Harm; Poulsen, Jens-Christian N.; Lo Leggio, Leila; Larsen, Sine; Christensen, Hans E. M.; Nagata, Kyosuke

2013-01-01

An N-terminally truncated mutant of the colicin E7 nuclease domain was crystallized and diffraction data set was collected to 1.6 Å resolution. The metallonuclease colicin E7 is a member of the HNH family of endonucleases. It serves as a bacterial toxin in Escherichia coli, protecting the host cell from other related bacteria and bacteriophages by degradation of their chromosomal DNA under environmental stress. Its cell-killing activity is attributed to the nonspecific nuclease domain (NColE7), which possesses the catalytic ββα-type metal ion-binding HNH motif at its C-terminus. Mutations affecting the positively charged amino acids at the N-terminus of NColE7 (444–576) surprisingly showed no or significantly reduced endonuclease activity [Czene et al. (2013 ▶), J. Biol. Inorg. Chem.18, 309–321]. The necessity of the N-terminal amino acids for the function of the C-terminal catalytic centre poses the possibility of allosteric activation within the enzyme. Precise knowledge of the intramolecular interactions of these residues that affect the catalytic activity could turn NColE7 into a novel platform for artificial nuclease design. In this study, the N-terminal deletion mutant ΔN4-NColE7-C* of the nuclease domain of colicin E7 selected by E. coli was overexpressed and crystallized at room temperature by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.6 Å resolution and could be indexed and averaged in the trigonal space group P3 1 21 or P3 2 21, with unit-cell parameters a = b = 55.4, c = 73.1 Å. Structure determination by molecular replacement is in progress

Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5' flap DNA: basis of interstrand cross-link repair by FAN1

Energy Technology Data Exchange (ETDEWEB)

Gwon, Gwang Hyeon; Kim, Youngran; Liu, Yaqi; Watson, Adam T.; Jo, Aera; Etheridge, Thomas J.; Yuan, Fenghua; Zhang, Yanbin; Kim, YoungChang; Carr, Anthony M.; Cho, Yunje

2014-10-15

Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of cross-links, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI–FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 5' flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domain playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 5' flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.

Revenue and Health Impacts of Restructuring Tobacco Excise Tax ...

International Development Research Centre (IDRC) Digital Library (Canada)

Revenue and Health Impacts of Restructuring Tobacco Excise Tax in the Philippines. A proposed law in the Philippines to increase the excise tax on tobacco by 215% will likely have implications for tobacco control and consumption, and public health, not just for that country but for the region. Although half of deaths due to ...

The adnAB Locus, Encoding a Putative Helicase-Nuclease Activity, Is Essential in Streptomyces

Science.gov (United States)

Zhang, Lingli; Nguyen, Hoang Chuong; Chipot, Ludovic; Piotrowski, Emilie; Bertrand, Claire

2014-01-01

Homologous recombination is a crucial mechanism that repairs a wide range of DNA lesions, including the most deleterious ones, double-strand breaks (DSBs). This multistep process is initiated by the resection of the broken DNA ends by a multisubunit helicase-nuclease complex exemplified by Escherichia coli RecBCD, Bacillus subtilis AddAB, and newly discovered Mycobacterium tuberculosis AdnAB. Here we show that in Streptomyces, neither recBCD nor addAB homologues could be detected. The only putative helicase-nuclease-encoding genes identified were homologous to M. tuberculosis adnAB genes. These genes are conserved as a single copy in all sequenced genomes of Streptomyces. The disruption of adnAB in Streptomyces ambofaciens and Streptomyces coelicolor could not be achieved unless an ectopic copy was provided, indicating that adnAB is essential for growth. Both adnA and adnB genes were shown to be inducible in response to DNA damage (mitomycin C) and to be independently transcribed. Introduction of S. ambofaciens adnAB genes in an E. coli recB mutant restored viability and resistance to UV light, suggesting that Streptomyces AdnAB could be a functional homologue of RecBCD and be involved in DNA damage resistance. PMID:24837284

Molecular cloning and characterization of genes required for nucleotide excision repair in yeast

International Nuclear Information System (INIS)

Friedberg, E.C.

1987-01-01

Nucleotide excision repair in the yeast S. cerevisiae is a complex process which involves a large number of genes. At least five of these genes (RAD1, RAD2, RAD3, RAD4 and RAD10) are absolutely required for this process and mutations in any of these genes result in no detectable excision repair in vivo. In order to understand the function of these genes in DNA repair, the authors isolated a number of them by screening a yeast genomic library for recombinant plasmids which complement the phentoype of sensitivity to ultraviolet (UV) radiation imparted to mutant strains. A plasmid containing the RAD4 gene was isolated by an alternative strategy which will be discussed. The cloned genes have been extensively characterized. It has been determined that the RAD3 gene is essential for the viability of haploid yeast cells in the absence of DNA damage. The RAD2 gene is inducible by treatment of cells with a variety of DNA-damaging agents, including UV radiation and ionizing radiation. The RAD10 gene shares considerable amino acid sequence homology with a cloned gene involved in nucleotide excision repair in human cells. Yeast is a particularly versatile organism for studying gene function by molecular and genetic approaches and emphasis is placed on many of the techniques used in the present studies

A Mismatch EndoNuclease Array-Based Methodology (MENA) for Identifying Known SNPs or Novel Point Mutations.

Science.gov (United States)

Comeron, Josep M; Reed, Jordan; Christie, Matthew; Jacobs, Julia S; Dierdorff, Jason; Eberl, Daniel F; Manak, J Robert

2016-04-05

Accurate and rapid identification or confirmation of single nucleotide polymorphisms (SNPs), point mutations and other human genomic variation facilitates understanding the genetic basis of disease. We have developed a new methodology (called MENA (Mismatch EndoNuclease Array)) pairing DNA mismatch endonuclease enzymology with tiling microarray hybridization in order to genotype both known point mutations (such as SNPs) as well as identify previously undiscovered point mutations and small indels. We show that our assay can rapidly genotype known SNPs in a human genomic DNA sample with 99% accuracy, in addition to identifying novel point mutations and small indels with a false discovery rate as low as 10%. Our technology provides a platform for a variety of applications, including: (1) genotyping known SNPs as well as confirming newly discovered SNPs from whole genome sequencing analyses; (2) identifying novel point mutations and indels in any genomic region from any organism for which genome sequence information is available; and (3) screening panels of genes associated with particular diseases and disorders in patient samples to identify causative mutations. As a proof of principle for using MENA to discover novel mutations, we report identification of a novel allele of the beethoven (btv) gene in Drosophila, which encodes a ciliary cytoplasmic dynein motor protein important for auditory mechanosensation.

Mung Bean nuclease mapping of RNAs 3' end

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Barbieri Rainer

2009-05-01

Full Text Available Abstract A method is described that allows an accurate mapping of 3' ends of RNAs. In this method a labeled DNA probe, containing the presumed 3' end of the RNA under analysis is allowed to anneals to the RNA itself. Mung-bean nuclease is then used to digest single strands of both RNA and DNA. Electrophoretic fractionation of "protected" undigested, labeled DNA is than performed using a sequence reaction of a known DNA as length marker. This procedure was applied to the analysis of both a polyA RNA (Interleukin 10 mRNA and non polyA RNAs (sea urchin 18S and 26S rRNAs. This method might be potentially relevant for the evaluation of the role of posttrascriptional control of IL-10 in the pathogenesis of the immune and inflammatory mediated diseases associated to ageing. This might allow to develop new strategies to approach to the diagnosis and therapy of age related diseases.

Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases

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Carmen F Bjurström

2016-01-01

Full Text Available We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs, transcriptional activator-like effector nucleases (TALENs, and clustered regularly interspaced short palindromic repeat (CRISPR/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA (pU6.g1 or in vitro transcribed gRNA (gR.1. Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs, highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs.

Excision of oral mucocele by different wavelength lasers

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Umberto Romeo

2013-01-01

Full Text Available Background: Mucocele is a common benign neoplasm of oral soft tissues and the most common after fibroma. It generally occurs in the lower lip and its treatment includes excision of cyst and the responsible salivary gland, in order to prevent recurrences. Aims: To evaluate the capability of three different lasers in performing the excision of labial mucocele with two different techniques. Materials and Methods: In the presented cases, excision was performed using two different techniques (circumferential incision technique and mucosal preservation technique and three different laser wavelengths (Er,Cr:YSGG 2780 nm, diode 808 nm, and KTP 532 nm. Results: All the tested lasers, regardless of wavelength, showed many advantages (bloodless surgical field, no postoperative pain, relative speed, and easy execution. The most useful surgical technique depends on clinical features of the lesion. Conclusion: Tested lasers, with both techniques, are helpful in the management of labial mucocele.

Recognition and repair of 2-aminofluorene- and 2-(acetylamino)fluorene-DNA adducts by UVRABC nuclease

International Nuclear Information System (INIS)

Pierce, J.R.; Case, R.; Tang, Moonshong

1989-01-01

Recognition of damage induced by N-hydroxy-2-aminofluorene (N-OH-AF) and N-acetoxy-2-(acetylamino)fluorene (NAAAF) in both φX174 RFI supercoiled DNA and a linear DNA fragment by purified UVRA, UVRB, and UVRC proteins was investigated. The authors have previously demonstrated that N-OH-AF and NAAAF treatments produce N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene (dG-C8-AAF), respectively, in DNA. Using a piperidine cleavage method and DNA sequence analysis, they have found that all guanine residues can be modified by N-OH-AF and NAAAF. These two kinds of adducts have different impacts on the DNA helix structure; while dG-C8-AF maintains the anti configuration, dG-C8-AAF is in the syn form. φX174 RF DNA-Escherichia coli transfection results indicate that while the uvrA, uvrB, and uvrC gene products are needed to repair dG-C8-AAF, the uvrC, but not the uvrA or uvrB gene products, is needed for repair of dG-C8-Af. However, they have found that in vitro the UVRA, UVRB, and UVRC proteins must work in concert to nick both dG-C8-AF and dG-C8-AAF. In general, the reactions of UVRABC nuclease toward dG-C8-AF are similar to those toward dG-C8-AAF; it incises seven to eight nucleotides from the 5' side and three to four nucleotides from the 3' side of the DNA adduct. Evidence is presented to suggest that hydrolysis on the 3' and 5' sides of the damaged base by UVRABC nuclease is not simultaneous and that at least occasionally hydrolysis occurs only on the 3' side or on the 5' side of the damage site. The possible mechanisms of UVRABC nuclease incision for AF-DNA are discussed

DNA excision repair in human cells treated with ultraviolet radiation and 7,12-dimethylbenz(a)anthracene 5,6-oxide

Energy Technology Data Exchange (ETDEWEB)

Ahmed, F.E.; Gentil, A.; Renstein, B.S.; Setlow, R.B.

1980-01-01

Excision repair was measured in normal human and xeroderma pigmentosum group C cells treated with 7,12-dimethylbenz(a)anthracene 5,6-oxide and with ultraviolet radiation by the techniques of unscheduled DNA synthesis, repair replication, a modification and bromodeoxyuridine photolysis and endonuclease-sensitive sites assay. Radiautography and repair replication showed that in normal cells the magnitude of repair after a saturation dose of the epoxide to be 0.1 to 0.2, that after a saturating ultraviolet dose, though survival data showed that both doses gave nearly similar killings. Repair was of the long-patch type and repair kinetics after the epoxide treatment were similar to ultraviolet. After a combined treatment with both agents, unscheduled synthesis in normal cells was more than additive. The data indicate that there are different rate-limiting steps in the removal of the ultraviolet and the epoxide damages, and that the residual repair activity in xeroderma pigmentosum cells is accomplished by different, not just fewer, enzymes than in normal cells.

Exposure of Human Lung Cells to Tobacco Smoke Condensate Inhibits the Nucleotide Excision Repair Pathway.

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Nathaniel Holcomb

Full Text Available Exposure to tobacco smoke is the number one risk factor for lung cancer. Although the DNA damaging properties of tobacco smoke have been well documented, relatively few studies have examined its effect on DNA repair pathways. This is especially true for the nucleotide excision repair (NER pathway which recognizes and removes many structurally diverse DNA lesions, including those introduced by chemical carcinogens present in tobacco smoke. The aim of the present study was to investigate the effect of tobacco smoke on NER in human lung cells. We studied the effect of cigarette smoke condensate (CSC, a surrogate for tobacco smoke, on the NER pathway in two different human lung cell lines; IMR-90 lung fibroblasts and BEAS-2B bronchial epithelial cells. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6-4 photoproducts and cyclobutane pyrimidine dimers. We find a dose-dependent inhibition of 6-4 photoproduct repair in both cell lines treated with CSC. Additionally, the impact of CSC on the abundance of various NER proteins and their respective RNAs was investigated. The abundance of XPC protein, which is required for functional NER, is significantly reduced by treatment with CSC while the abundance of XPA protein, also required for NER, is unaffected. Both XPC and XPA RNA levels are modestly reduced by CSC treatment. Finally, treatment of cells with MG-132 abrogates the reduction in the abundance of XPC protein produced by treatment with CSC, suggesting that CSC enhances proteasome-dependent turnover of the protein that is mediated by ubiquitination. Together, these findings indicate that tobacco smoke can inhibit the same DNA repair pathway that is also essential for the removal of some of the carcinogenic DNA damage introduced by smoke itself, increasing the DNA damage burden of cells exposed to tobacco smoke.

Recovery of DNA synthesis after ultraviolet irradiation of xeroderma pigmentosum cells depends on excision repair and is blocked by caffeine

International Nuclear Information System (INIS)

Park, S.D.; Cleaver, J.E.

1979-01-01

Normal human and xeroderma pigmentosum (XP, excision-defective group A) cells (both SV40-transformed) pulse-labeled with [ 3 H] thymidine at various times after irradiation with ultraviolet light showed a decline and recovery of both the molecular weights of newly synthesized DNA and the rated of synthesis per cell. At the same ultraviolet dose, both molecular weights and rates of synthesis were inhibited more in XP than in normal cells. This indicates that excision repair plays a role in minimizing the inhibition of chain growth, possibly by excision of dimers ahead of the growing point. The ability to synthesize normal-sized DNA recovered more rapidly than rates of synthesis in normal cells, but both parameters recovered in phase in XP cells. During recovery in normal cells there are therefore fewer actively replicating clusters of replicons because the single-strand breaks involved in the excision of dimers inhibit replicon initiation. XP cells have few excision repair events and therefore fewer breaks to interfere with initiation, but chain growth is blocked by unexcised dimers. In both cell types recovery of the ability to synthesize normal-sized DNA was prevented by growing cells in caffeine after irradiation, possibly because of competition between the DNA binding properties of caffeine and replication proteins. These observations imply that excision repair and semiconservative replication interact strongly in irradiated cells to produce a complex spectrum of changes in DNA replication which may be confused with parts of alternative systems such as post-replication repair. (author)

Effects of burn wound excision on bacterial colonization and invasion

NARCIS (Netherlands)

Barret, JP; Herndon, DN

Rates of survival after thermal injury have improved in the past two decades, and rates of wound infections and sepsis have decreased during the same period. Early excision has been advocated as one of the major factors, but its safety and efficacy and the exact timing of burn excision are still

Excision of Mucocele Using Diode Laser in Lower Lip

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Subramaniam Ramkumar

2016-01-01

Full Text Available Mucoceles are nonneoplastic cystic lesions of major and minor salivary glands which result from the accumulation of mucus. These lesions are most commonly seen in children. Though usually these lesions can be treated by local surgical excision, in our case, to avoid intraoperative surgical complications like bleeding and edema and to enable better healing, excision was done using a diode laser in the wavelength of 940 nm.

Coincident resection at both ends of random, γ-induced double-strand breaks requires MRX (MRN, Sae2 (Ctp1, and Mre11-nuclease.

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James W Westmoreland

2013-03-01

Full Text Available Resection is an early step in homology-directed recombinational repair (HDRR of DNA double-strand breaks (DSBs. Resection enables strand invasion as well as reannealing following DNA synthesis across a DSB to assure efficient HDRR. While resection of only one end could result in genome instability, it has not been feasible to address events at both ends of a DSB, or to distinguish 1- versus 2-end resections at random, radiation-induced "dirty" DSBs or even enzyme-induced "clean" DSBs. Previously, we quantitatively addressed resection and the role of Mre11/Rad50/Xrs2 complex (MRX at random DSBs in circular chromosomes within budding yeast based on reduced pulsed-field gel electrophoretic mobility ("PFGE-shift". Here, we extend PFGE analysis to a second dimension and demonstrate unique patterns associated with 0-, 1-, and 2-end resections at DSBs, providing opportunities to examine coincidence of resection. In G2-arrested WT, Δrad51 and Δrad52 cells deficient in late stages of HDRR, resection occurs at both ends of γ-DSBs. However, for radiation-induced and I-SceI-induced DSBs, 1-end resections predominate in MRX (MRN null mutants with or without Ku70. Surprisingly, Sae2 (Ctp1/CtIP and Mre11 nuclease-deficient mutants have similar responses, although there is less impact on repair. Thus, we provide direct molecular characterization of coincident resection at random, radiation-induced DSBs and show that rapid and coincident initiation of resection at γ-DSBs requires MRX, Sae2 protein, and Mre11 nuclease. Structural features of MRX complex are consistent with coincident resection being due to an ability to interact with both DSB ends to directly coordinate resection. Interestingly, coincident resection at clean I-SceI-induced breaks is much less dependent on Mre11 nuclease or Sae2, contrary to a strong dependence on MRX complex, suggesting different roles for these functions at "dirty" and clean DSB ends. These approaches apply to resection at

Nucleotide excision repair- and p53-deficient mouse models in cancer research

Energy Technology Data Exchange (ETDEWEB)

Hoogervorst, Esther M. [Laboratory of Toxicology, Pathology and Genetics, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven (Netherlands); Utrecht University, Department of Pathobiology, Utrecht (Netherlands); Steeg, Harry van [Laboratory of Toxicology, Pathology and Genetics, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven (Netherlands); Vries, Annemieke de [Laboratory of Toxicology, Pathology and Genetics, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven (Netherlands)]. E-mail: Annemieke.de.Vries@rivm.nl

2005-07-01

Cancer is caused by the loss of controlled cell growth due to mutational (in)activation of critical genes known to be involved in cell cycle regulation. Three main mechanisms are known to be involved in the prevention of cells from becoming cancerous; DNA repair and cell cycle control, important to remove DNA damage before it will be fixed into mutations and apoptosis, resulting in the elimination of cells containing severe DNA damage. Several human syndromes are known to have (partially) deficiencies in these pathways, and are therefore highly cancer prone. Examples are xeroderma pigmentosum (XP) caused by an inborn defect in the nucleotide excision repair (NER) pathway and the Li-Fraumeni syndrome, which is the result of a germ line mutation in the p53 gene. XP patients develop skin cancer on sun exposed areas at a relatively early age, whereas Li-Fraumeni patients spontaneously develop a wide variety of early onset tumors, including sarcomas, leukemia's and mammary gland carcinomas. Several mouse models have been generated to mimic these human syndromes, providing us information about the role of these particular gene defects in the tumorigenesis process. In this review, spontaneous phenotypes of mice deficient for nucleotide excision repair and/or the p53 gene will be described, together with their responses upon exposure to either chemical carcinogens or radiation. Furthermore, possible applications of these and newly generated mouse models for cancer will be given.

Re-excision rates after breast conserving surgery following the 2014 SSO-ASTRO guidelines.

Science.gov (United States)

Heelan Gladden, Alicia A; Sams, Sharon; Gleisner, Ana; Finlayson, Christina; Kounalakis, Nicole; Hosokawa, Patrick; Brown, Regina; Chong, Tae; Mathes, David; Murphy, Colleen

2017-12-01

In 2014, SSO-ASTRO published guidelines which recommended "no ink on tumor" as adequate margins for patients undergoing breast conservation for invasive breast cancer. In 2016, new SSO-ASTRO-ASCO guidelines recommended 2 mm margins for DCIS. We evaluated whether these guidelines affected re-excision rates at our institution. Patients treated with breast conservation surgery from January 1, 2010-March 1, 2016 were identified. Re-excision rates, tumor characteristics, and presence of residual disease were recorded. The 2016 guidelines were retrospectively applied to the same cohort and expected re-excision rates calculated. Re-excision rates did not significantly decline before and after 2014 guideline adoption (11.9% before, 10.9% after; p = 0.65) or when the 2016 guidelines were retrospectively applied (8.4%; p = 0.10). The 2014 and 2016 guidelines had minimal impact on our re-excision rates, as most re-excisions were done for DCIS and 2016 guidelines supported our prior institutional practices of 2 mm margins for these patients. Copyright © 2017 Elsevier Inc. All rights reserved.

Use of zinc-finger nucleases to knock out the WAS gene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome

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Miguel G. Toscano

2013-03-01

Mutations in the WAS gene cause Wiskott-Aldrich syndrome (WAS, which is characterized by eczema, immunodeficiency and microthrombocytopenia. Although the role of WASP in lymphocytes and myeloid cells is well characterized, its role on megakaryocyte (MK development is poorly understood. In order to develop a human cellular model that mimics the megakaryocytic-derived defects observed in WAS patients we used K562 cells, a well-known model for study of megakaryocytic development. We knocked out the WAS gene in K562 cells using a zinc-finger nuclease (ZFN pair targeting the WAS intron 1 and a homologous donor DNA that disrupted WASP expression. Knockout of WASP on K562 cells (K562WASKO cells resulted in several megakaryocytic-related defects such as morphological alterations, lower expression of CD41ɑ, lower increments in F-actin polymerization upon stimulation, reduced CD43 expression and increased phosphatidylserine exposure. All these defects have been previously described either in WAS-knockout mice or in WAS patients, validating K562WASKO as a cell model for WAS. However, K562WASPKO cells showed also increased basal F-actin and adhesion, increased expression of CD61 and reduced expression of TGFβ and Factor VIII, defects that have never been described before for WAS-deficient cells. Interestingly, these phenotypic alterations correlate with different roles for WASP in megakaryocytic differentiation. All phenotypic alterations observed in K562WASKO cells were alleviated upon expression of WAS following lentiviral transduction, confirming the role of WASP in these phenotypes. In summary, in this work we have validated a human cellular model, K562WASPKO, that mimics the megakaryocytic-related defects found in WAS-knockout mice and have found evidences for a role of WASP as regulator of megakaryocytic differentiation. We propose the use of K562WASPKO cells as a tool to study the molecular mechanisms involved in the megakaryocytic-related defects observed in WAS

Optomagnetic Detection of MicroRNA Based on Duplex-Specific Nuclease-Assisted Target Recycling and Multilayer Core-Satellite Magnetic Superstructures

DEFF Research Database (Denmark)

Tian, Bo; Ma, Jing; Qiu, Zhen

2017-01-01

-efficiency, and potential for bioresponsive multiplexing. Herein, we demonstrate a sensitive and rapid miRNA detection method based on optomagnetic read-out, duplex-specific nuclease (DSN)-assisted target recycling, and the use of multilayer core-satellite magnetic superstructures. Triggered by the presence of target mi...

Beyond Wires and Seeds: Reflector-guided Breast Lesion Localization and Excision.

Science.gov (United States)

Mango, Victoria L; Wynn, Ralph T; Feldman, Sheldon; Friedlander, Lauren; Desperito, Elise; Patel, Sejal N; Gomberawalla, Ameer; Ha, Richard

2017-08-01

Purpose To evaluate outcomes of Savi Scout (Cianna Medical, Aliso Viejo, Calif) reflector-guided localization and excision of breast lesions by analyzing reflector placement, localization, and removal, along with target excision and rates of repeat excision (referred to as re-excision). Materials and Methods A single-institution retrospective review of 100 women who underwent breast lesion localization and excision by using the Savi Scout surgical guidance system from June 2015 to May 2016 was performed. By using image guidance 0-8 days before surgery, 123 nonradioactive, infrared-activated, electromagnetic wave reflectors were percutaneously inserted adjacent to or within 111 breast targets. Twenty patients had two or three reflectors placed for bracketing or for localizing multiple lesions, and when ipsilateral, they were placed as close as 2.6 cm apart. Target and reflector were localized intraoperatively by one of two breast surgeons who used a handpiece that emitted infrared light and electromagnetic waves. Radiographs of the specimen and pathologic analysis helped verify target and reflector removal. Target to reflector distance was measured on the mammogram and radiograph of the specimen, and reflector depth was measured on the mammogram. Pathologic analysis was reviewed. Re-excision rates and complications were recorded. By using statistics software, descriptive statistics were generated with 95% confidence intervals (CIs) calculated. Results By using sonographic (40 of 123; 32.5%; 95% CI: 24.9%, 41.2%) or mammographic (83 of 123; 67.5%; 95% CI: 58.8% 75.1%) guidance, 123 (100%; 95% CI: 96.4%, 100%) reflectors were placed. Mean mammographic target to reflector distance was 0.3 cm. All 123 (100%; 95% CI: 96.4%, 100%) targets and reflectors were excised. Pathologic analysis yielded 54 of 110 malignancies (49.1%; 95% CI: 39.9%, 58.3%; average, 1.0 cm; range, 0.1-5 cm), 32 high-risk lesions (29.1%; 95% CI: 21.4%, 38.2%), and 24 benign lesions (21.8%; 95% CI

Total excision of intramedullary epidermoid cyst in one case

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PU Ke

2013-12-01

Full Text Available Clinical experience of total excision in a 14-year-old female with intramedullary epidermoid cyst was reported. The patient with L3-4 intramedullary epidermoid cyst underwent total excision through posterior median approach under microscopy. The patient was admitted for progressive strephexopodia and urinary and fecal incontinence. Preoperative imaging examination showed scoliosis, incontionous L4-S1 and abnormal signal of L3-4. Total excision and spinal remodeling were performed under intraoperative neurophysiological monitoning. Epidermoid cyst and its membrane were totally removed without aseptic meningitis after surgery, and the neurologic symptoms of the patient were gradually improved. Completely removing the membrane of epidermoid cyst is the key point to prevent recurrence and aseptic meningitis postoperatively. Dissection should be strictly in accordance with the boundaries of the membrane and the spinal cord, in order to avoid spinal cord injury.

Resolution of vitiligo following excision of halo congenital melanocytic nevus: a rare case report.

Science.gov (United States)

Wang, Kai; Wang, Zhi; Huang, Weiqing

2016-05-01

Halo congenital melanocytic nevus (CMN) associated with vitiligo is rare, especially with regard to CMN excision. Only two reports of excision of halo CMN following repigmentation of vitiligo are found in the literature. We present a case of a girl with halo CMN and periorbital vitiligo. The halo CMN was excised and followed by spontaneous improvement of vitiligo. The result suggests excision of the inciting lesion may be a promising way to control vitiligo. © 2015 Wiley Periodicals, Inc.

Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells.

Science.gov (United States)

Al-Serori, Halh; Ferk, Franziska; Kundi, Michael; Bileck, Andrea; Gerner, Christopher; Mišík, Miroslav; Nersesyan, Armen; Waldherr, Monika; Murbach, Manuel; Lah, Tamara T; Herold-Mende, Christel; Collins, Andrew R; Knasmüller, Siegfried

2018-01-01

Some epidemiological studies indicate that the use of mobile phones causes cancer in humans (in particular glioblastomas). It is known that DNA damage plays a key role in malignant transformation; therefore, we investigated the impact of the UMTS signal which is widely used in mobile telecommunications, on DNA stability in ten different human cell lines (six brain derived cell lines, lymphocytes, fibroblasts, liver and buccal tissue derived cells) under conditions relevant for users (SAR 0.25 to 1.00 W/kg). We found no evidence for induction of damage in single cell gel electrophoresis assays when the cells were cultivated with serum. However, clear positive effects were seen in a p53 proficient glioblastoma line (U87) when the cells were grown under serum free conditions, while no effects were found in p53 deficient glioblastoma cells (U251). Further experiments showed that the damage disappears rapidly in U87 and that exposure induced nucleotide excision repair (NER) and does not cause double strand breaks (DSBs). The observation of NER induction is supported by results of a proteome analysis indicating that several proteins involved in NER are up-regulated after exposure to UMTS; additionally, we found limited evidence for the activation of the γ-interferon pathway. The present findings show that the signal causes transient genetic instability in glioma derived cells and activates cellular defense systems.

Excision of a viral reprogramming cassette by delivery of synthetic Cre mRNA

Science.gov (United States)

Loh, Yuin-Han; Yang, Jimmy Chen; De Los Angeles, Alejandro; Guo, Chunguang; Cherry, Anne; Rossi, Derrick J.; Park, In-Hyun; Daley, George Q.

2012-01-01

The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy, in vitro modeling of human disease, and drug screening. To date, most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively, transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This report describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector, which includes the coding sequences of human OCT4, SOX2, KLF4, and cMYC linked with picornaviral 2A plasmids. Moreover, after reprogramming has been achieved, this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells. PMID:22605648

Modeling of 5 ' nuclease real-time responses for optimization of a high-throughput enrichment PCR procedure for Salmonella enterica

DEFF Research Database (Denmark)

Knutsson, R.; Löfström, Charlotta; Grage, H.

2002-01-01

The performance of a 5' nuclease real-time PCR assay was studied to optimize an automated method of detection of preenriched Salmonella enterica cells in buffered peptone water (BPW). The concentrations and interactions of the PCR reagents were evaluated on the basis of two detection responses, t...

Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID using zinc-finger nucleases.

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Tomoji Mashimo

Full Text Available BACKGROUND: Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc. METHODOLOGY/PRINCIPAL FINDINGS: We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID. Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype. CONCLUSIONS AND SIGNIFICANCE: The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.

Circumareolar incision-subdermal tunneling dissection for excision of multiple breast fibroadenomata

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S O Agodirin

2017-01-01

Full Text Available Excision of multiple fibroadenomas (MF in separate breast quadrants presents difficulties of number and location of incision(s and extent of tissue dissection and may be associated with more complications and poorer cosmetic outcome. This is a report of excision of MF in multiple quadrants of the breast using a modification of subcutaneous dissection technique dubbed the circumareolar incision and subdermal tunneling (CAST dissection. After exposure of the superficial fascia with circumareolar incision, subdermal cone-wise dissection was made to allow mobilization of the segment bearing the lump(s. The lump(s were enucleated and removed. MF were removed from four breasts in three young unmarried females. The first patient had multiple adenomas removed from three quadrants of both breasts: 14 on the right and six on the left. The second patient had excision of three lumps in three separate quadrants, and the third patient had excision of two lumps in two separate quadrants. All patients had edema and bruising. One breast had wound infection and dehiscence. There were no skin necrosis, no nipple loss, and no breast distortion. All ensuing scars were camouflaged. CAST dissection was used for excision of MF in multiple quadrants of the breast with preservation of excellent cosmetic outcome of a single circumareolar incision.

mei-9/sup a/ mutant of Drosophila melanogaster increases mutagen sensitivity and decreases excision repair

International Nuclear Information System (INIS)

Boyd, J.B.; Golino, M.D.; Setlow, R.B.

1976-01-01

The mei-9/sup a/ mutant of Drosophila melanogaster, which reduces meiotic recombination in females, is deficient in the excision of uv-induced pyrimidine dimers in both sexes. Assays were performed in primary cultures and established cell lines derived from embryos. An endonuclease preparation from M. luteus, which is specific for pyrimidine dimers, was employed to monitor uv-induced dimers in cellular DNA. The rate of disappearance of endonuclease-sensitive sites from DNA of control cells is 10-20 times faster than that from mei-9/sup a/ cells. The mutant mei-218, which is also deficient in meiotic recombination, removes nuclease-sensitive sites at control rates. The mei-9/sup a/ cells exhibit control levels of photorepair, postreplication repair and repair of single strand breaks. In mei-9 cells DNA synthesis and possibly postreplication repair are weakly sensitive to caffeine. Larvae which are hemizygous for either of the two mutants that define the mei-9 locus are hypersensitive to killing by the mutagens methyl methanesulfonate, nitrogen mustard and 2-acetylaminofluorene. Larvae hemizygous for the mei-218 mutant are insensitive to each of these reagents. These data demonstrate that the mei-9 locus is active in DNA repair of somatic cells. Thus functions involved in meiotic recombination are also active in DNA repair in this higher eukaryote. The results are consistent with the earlier suggestions that the mei-9 locus functions in the exchange events of meiosis. The mei-218 mutation behaves differently in genetic tests and our data suggest its function may be restricted to meiosis. These studies demonstrate that currently recognized modes of DNA repair can be efficiently detected in primary cell cultures derived from Drosophila embryos

Nucleotide excision repair in the test tube.

NARCIS (Netherlands)

N.G.J. Jaspers (Nicolaas); J.H.J. Hoeijmakers (Jan)

1995-01-01

textabstractThe eukaryotic nucleotide excision-repair pathway has been reconstituted in vitro, an achievement that should hasten the full enzymological characterization of this highly complex DNA-repair pathway.

Excision technique in constrained formulations of Einstein equations: collapse scenario

International Nuclear Information System (INIS)

Cordero-Carrión, I; Vasset, N; Novak, J; Jaramillo, J L

2015-01-01

We present a new excision technique used in constrained formulations of Einstein equations to deal with black hole in numerical simulations. We show the applicability of this scheme in several scenarios. In particular, we present the dynamical evolution of the collapse of a neutron star to a black hole, using the CoCoNuT code and this excision technique. (paper)

Obturator foramen dissection for excision of symptomatic transobturator mesh.

Science.gov (United States)

Reynolds, W Stuart; Kit, Laura Chang; Kaufman, Melissa R; Karram, Mickey; Bales, Gregory T; Dmochowski, Roger R

2012-05-01

Groin pain after transobturator synthetic mesh placement can be recalcitrant to conservative therapy and ultimately requires surgical excision. We describe our experiences with and technique of obturator foramen dissection for mesh excision. The records of 8 patients treated from 2005 to 2010, were reviewed. Obturator dissection was performed via a lateral groin incision over the inferior pubic ramus at the level of the obturator foramen, typically in conjunction with orthopedic surgery. Five patients had transobturator mid urethral sling surgery for stress urinary incontinence, 2 had mid urethral sling and trocar based anterior vaginal wall mesh kits with transobturator passage of mesh arms for stress urinary incontinence and pelvic organ prolapse, and 1 had an anterior vaginal wall mesh kit for pelvic organ prolapse. Patients had 0 to 2 prior transvaginal mesh excisions before obturator surgery. All patients presented with intractable pain in the area of the obturator foramen and/or medial groin for which conservative treatment measures had failed. Six patients underwent concurrent vaginal and obturator dissection and 2 underwent obturator dissection alone. In all cases residual mesh (3 to 11 cm) was identified and excised from the obturator foramen. Mesh was closely associated to or traversing the adductor longus muscle and tendon with significant fibrous reaction in all cases. Postoperatively 5 patients were cured of pain and/or infection, and 3 reported no or some improvement at a mean followup of 6 months (range 1 to 12). Our experience suggests that surgical excision of residual mesh can alleviate many of the symptoms in many patients. In all cases mesh remnants were identified and removed, and typically involved neuromuscular structures adjacent to the obturator foramen. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

The effect of excise tax increases on cigarette prices in South Africa

Science.gov (United States)

Linegar, Daniel J; van Walbeek, Corne

2018-01-01

Introduction The effectiveness of excise tax increases as a tool for reducing tobacco consumption depends largely on how the tax increases impact the retail price. We estimate this relationship in South Africa for 2001–2015. Data Statistics South Africa provided disaggregated cigarette price data, used in the calculation of the Consumers’ Price Index. Data on the excise tax per cigarette were obtained from Budget Reviews prepared by the National Treasury of South Africa. Methods Regression equations were estimated for each month. The month-on-month change in cigarette prices in February through April was regressed against March’s excise tax change to estimate the pass-through coefficient. For the other 9 months, the month-on-month change in cigarette price was regressed against monthly dummy variables to determine the size of the non-tax-related price increase in each of these months. The analysis was performed in both nominal and real (inflation-adjusted) terms. Findings Expressed in real terms, the excise tax was undershifted. A R1.00 (one rand) increase in the excise tax is associated with an increase in the retail price of cigarettes of R0.90 in the pre-2010 period, and R0.49 in the post-2010 period. In the pre-2010 period, the tobacco industry increased the retail price of cigarettes in July/August, independent of the excise tax increase. The discretionary July/August price increases largely disappeared after 2010, primarily because the market became more competitive. Conclusion The degree of excise tax pass-through, and the magnitude of discretionary increases in cigarette prices, is significantly determined by the competitive environment in the cigarette market. PMID:28341767

Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

International Nuclear Information System (INIS)

Watanabe, Masahito; Umeyama, Kazuhiro; Matsunari, Hitomi; Takayanagi, Shuko; Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka; Nakauchi, Hiromitsu

2010-01-01

Research highlights: → EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. → ZFNs induced targeted mutations in porcine primary cultured cells. → Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

Accurate DNA assembly and genome engineering with optimized uracil excision cloning

DEFF Research Database (Denmark)

Cavaleiro, Mafalda; Kim, Se Hyeuk; Seppala, Susanna

2015-01-01

Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway that pro......Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway...... that produces β-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly...... facilitating the advanced engineering of robust cell factories....

Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation

Energy Technology Data Exchange (ETDEWEB)

Konze-Thomas, B.; Hazard, R.M.; Maher, V.M.; McCormick, J.J. (Michigan State Univ., East Lansing (USA). Carcinogenesis Lab.)

1982-01-01

Excision repair-proficient diploid fibroblasts from normal persons (NF) and repair-deficient cells from a xeroderma pigmentosum patient (XP12BE, group A) were grown to confluence and allowed to enter the G/sub 0/ state. Autoradiography studies of cells released from G/sub 0/ after 72 h and replated at lower densities (3-9 x 10/sup 3/ cells/cm/sup 2/) in fresh medium showed that semiconservative DNA synthesis (S phase) began approx. equal to 24 h after the replating. The task was to determine whether the time available for DNA excision repair between ultraviolet irradiation (254 nm) and the onset of DNA synthesis was critical in determining the cytotoxic and/or mutagenic effect of UV in human fibroblasts.

Pulsed terahertz imaging of breast cancer in freshly excised murine tumors

Science.gov (United States)

Bowman, Tyler; Chavez, Tanny; Khan, Kamrul; Wu, Jingxian; Chakraborty, Avishek; Rajaram, Narasimhan; Bailey, Keith; El-Shenawee, Magda

2018-02-01

This paper investigates terahertz (THz) imaging and classification of freshly excised murine xenograft breast cancer tumors. These tumors are grown via injection of E0771 breast adenocarcinoma cells into the flank of mice maintained on high-fat diet. Within 1 h of excision, the tumor and adjacent tissues are imaged using a pulsed THz system in the reflection mode. The THz images are classified using a statistical Bayesian mixture model with unsupervised and supervised approaches. Correlation with digitized pathology images is conducted using classification images assigned by a modal class decision rule. The corresponding receiver operating characteristic curves are obtained based on the classification results. A total of 13 tumor samples obtained from 9 tumors are investigated. The results show good correlation of THz images with pathology results in all samples of cancer and fat tissues. For tumor samples of cancer, fat, and muscle tissues, THz images show reasonable correlation with pathology where the primary challenge lies in the overlapping dielectric properties of cancer and muscle tissues. The use of a supervised regression approach shows improvement in the classification images although not consistently in all tissue regions. Advancing THz imaging of breast tumors from mice and the development of accurate statistical models will ultimately progress the technique for the assessment of human breast tumor margins.

Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases.

Science.gov (United States)

Citorik, Robert J; Mimee, Mark; Lu, Timothy K

2014-11-01

Current antibiotics tend to be broad spectrum, leading to indiscriminate killing of commensal bacteria and accelerated evolution of drug resistance. Here, we use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design. RNA-guided nucleases (RGNs) targeting specific DNA sequences are delivered efficiently to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation. The DNA targets of RGNs can be undesirable genes or polymorphisms, including antibiotic resistance and virulence determinants in carbapenem-resistant Enterobacteriaceae and enterohemorrhagic Escherichia coli. Delivery of RGNs significantly improves survival in a Galleria mellonella infection model. We also show that RGNs enable modulation of complex bacterial populations by selective knockdown of targeted strains based on genetic signatures. RGNs constitute a class of highly discriminatory, customizable antimicrobials that enact selective pressure at the DNA level to reduce the prevalence of undesired genes, minimize off-target effects and enable programmable remodeling of microbiota.

Comparison between preoperative biopsy and post-excision ...

African Journals Online (AJOL)

Comparison between preoperative biopsy and post-excision histology results in sarcoma: Experience at Chris Hani Baragwanath Academic Hospital, Johannesburg, South Africa. KG Panda, MJ Hale, D Kruger, TE Luvhengo ...

Global-genome Nucleotide Excision Repair Controlled by Ubiquitin/Sumo Modifiers

Directory of Open Access Journals (Sweden)

Peter eRuethemann

2016-04-01

Full Text Available Global-genome nucleotide excision repair (GG-NER prevents genome instability by excising a wide range of structurally unrelated DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV radiation or intracellular metabolic by-products. As a versatile damage sensor, xeroderma pigmentosum group C (XPC protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4DDB2 and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4DDB2 or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin.

New insights in the removal of the hydantoins, oxidation product of pyrimidines, via the base excision and nucleotide incision repair pathways.

Directory of Open Access Journals (Sweden)

Modesto Redrejo-Rodríguez

Full Text Available BACKGROUND: Oxidative damage to DNA, if not repaired, can be both miscoding and blocking. These genetic alterations can lead to mutations and/or cell death, which in turn cause cancer and aging. Oxidized DNA bases are substrates for two overlapping repair pathways: base excision (BER and nucleotide incision repair (NIR. Hydantoin derivatives such as 5-hydroxyhydantoin (5OH-Hyd and 5-methyl-5-hydroxyhydantoin (5OH-5Me-Hyd, major products of cytosine and thymine oxidative degradation pathways, respectively, have been detected in cancer cells and ancient DNA. Hydantoins are blocking lesions for DNA polymerases and excised by bacterial and yeast DNA glycosylases in the BER pathway. However little is known about repair of pyrimidine-derived hydantoins in human cells. METHODOLOGY/PRINCIPAL FINDINGS: Here, using both denaturing PAGE and MALDI-TOF MS analyses we report that the bacterial, yeast and human AP endonucleases can incise duplex DNA 5' next to 5OH-Hyd and 5OH-5Me-Hyd thus initiating the NIR pathway. We have fully reconstituted the NIR pathway for these lesions in vitro using purified human proteins. Depletion of Nfo in E. coli and APE1 in HeLa cells abolishes the NIR activity in cell-free extracts. Importantly, a number of redundant DNA glycosylase activities can excise hydantoin residues, including human NTH1, NEIL1 and NEIL2 and the former protein being a major DNA glycosylase activity in HeLa cells extracts. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that both BER and NIR pathways can compete and/or back-up each other to remove hydantoin DNA lesions in vivo.

Quantitative characterization of pyrimidine dimer excision from UV-irradiated DNA (excision capacity) by cell-free extracts of the yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Bekker, M.L.; Kaboev, O.K.; Akhmedov, A.T.; Luchkina, L.A.

1984-01-01

Cell-free extracts from wild-type yeast (RAD + ) and from rad mutants belonging to the RAD3 epistatic group (rad1-1, rad2-1, rad3-1, rad4-1) contain activities catalyzing the excision of pyrimidine dimers (PD) from purified ultraviolet-irradiated DNA which was not pre-treated with exogenous UV-endonuclease. The level of these activities in cell-free extracts from rad mutants did not differ from that in wild-type extract and was close to the in vivo excision capacity of the latter calculated from the LD 37 (about 10 4 PD per haploid genome). (Auth.)

Excised Abdominoplasty Material as a Systematic Plastic Surgical Training Model

Directory of Open Access Journals (Sweden)

M. Erol Demirseren

2012-01-01

Full Text Available Achieving a level of technical skill and confidence in surgical operations is the main goal of plastic surgical training. Operating rooms were accepted as the practical teaching venues of the traditional apprenticeship model. However, increased patient population, time, and ethical and legal considerations made preoperation room practical work a must for plastic surgical training. There are several plastic surgical teaching models and simulators which are very useful in preoperation room practical training and the evaluation of plastic surgery residents. The full thickness skin with its vascular network excised in abdominoplasty procedures is an easily obtainable real human tissue which could be used as a training model in plastic surgery.

Genome Editing: A New Approach to Human Therapeutics.

Science.gov (United States)

Porteus, Matthew

2016-01-01

The ability to manipulate the genome with precise spatial and nucleotide resolution (genome editing) has been a powerful research tool. In the past decade, the tools and expertise for using genome editing in human somatic cells and pluripotent cells have increased to such an extent that the approach is now being developed widely as a strategy to treat human disease. The fundamental process depends on creating a site-specific DNA double-strand break (DSB) in the genome and then allowing the cell's endogenous DSB repair machinery to fix the break such that precise nucleotide changes are made to the DNA sequence. With the development and discovery of several different nuclease platforms and increasing knowledge of the parameters affecting different genome editing outcomes, genome editing frequencies now reach therapeutic relevance for a wide variety of diseases. Moreover, there is a series of complementary approaches to assessing the safety and toxicity of any genome editing process, irrespective of the underlying nuclease used. Finally, the development of genome editing has raised the issue of whether it should be used to engineer the human germline. Although such an approach could clearly prevent the birth of people with devastating and destructive genetic diseases, questions remain about whether human society is morally responsible enough to use this tool.

Involvement of UV-inducible repair in pyrimidine dimer excision in Escherichia coli

International Nuclear Information System (INIS)

Masek, F.; Sedliakova, M.

1978-01-01

The influence of UV radiation on pyrimidine dimer excision in the cells of three excision-proficient E.coli strains was studied. For this purpose cells were irradiated with a first fluence of 300 ergs/mm 2 and at different time intervals with a second fluence of 500 ergs/mm 2 . After the second fluence dimer excision was found to be partly inhibited in E.coli B/r Hcr + and E.coli 15 555-7, but not in E.coli K12 SR20. (author)

Involvement of UV-inducible repair in pyrimidine dimer excision in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Masek, F; Sedliakova, M [Slovenska Akademia Vied, Bratislava (Czechoslovakia)

1978-11-15

The influence of UV radiation on pyrimidine dimer excision in the cells of three excision-proficient E.coli strains was studied. For this purpose cells were irradiated with a first fluence of 300 ergs/mm/sup 2/ and at different time intervals with a second fluence of 500 ergs/mm/sup 2/. After the second fluence dimer excision was found to be partly inhibited in E.coli B/r Hcr/sup +/ and E.coli 15 555-7, but not in E.coli K12 SR20.

S1-sensitive sites in DNA after γ-irradiation

International Nuclear Information System (INIS)

Martin-Bertram, H.

1981-01-01

DNA from γ-irradiated T 1 bacteriophages was analyzed for 'single-stranded' sites by cleavage with S1 nuclease from Aspergillus oryzae as lesion probe. The ratio of 'S1-sensitive sites' to the amount of radiation-induced single-strand breaks was about one. Presumably these 'denatured' sites were associated with single-strand breaks. The subsequent check for the persistence of 'single-stranded' sites within the DNA molecule by thermokinetics demonstrated a strong affinity of the nuclease to its substrate, the single-stranded lesion, and a perfect excision. It is assumed that the direct absorption of radiation energy in the DNA gives rise to the formation of such bulky lesions. (Auth.)

Frequency and outcomes of biopsy-proven fibroadenomas recommended for surgical excision.

Science.gov (United States)

Lee, Shimwoo; Mercado, Cecilia L; Cangiarella, Joan F; Chhor, Chloe M

2017-12-16

Our aim was to investigate the outcomes of fibroadenomas recommended for surgical excision due to large size (>2cm) or interval growth. A retrospective review of our institutional radiology database from 2007 to 2015 was performed. We identified 167 biopsy-proven fibroadenomas recommended for surgical consultation. Of these, 75 (45%) cases actually underwent excision, 7 (9%, 95% CI: 4-18%) of which were upgraded to phyllodes tumors upon histopathological examination. Our results support the current recommendation to surgically excise breast lesions diagnosed as fibroadenomas with size >2cm or with interval growth due to the considerable risk of finding phyllodes tumors. Copyright © 2017 Elsevier Inc. All rights reserved.

CELL POPULATION KINETICS OF EXCISED ROOTS OF PISUM SATIVUM

Science.gov (United States)

Van't Hof, Jack

1965-01-01

The cell population kinetics of excised, cultured pea roots was studied with the use of tritiated thymidine and colchicine to determine (1) the influence of excision, (2) the influence of sucrose concentration, (3) the average mitotic cycle duration, and (4) the duration of mitosis and the G 1, S, and G 2 periods of interphase.1 The results indicate that the process of excision causes a drop in the frequency of mitotic figures when performed either at the beginning of the culture period or after 100 hours in culture. This initial decrease in frequency of cell division is independent of sucrose concentration, but the subsequent rise in frequency of division, after 12 hours in culture, is dependent upon sucrose concentration. Two per cent sucrose maintains the shortest mitotic cycle duration. The use of colchicine indicated an average cycle duration of 20 hours, whereas the use of tritiated thymidine produced an average cycle duration of 17 hours. PMID:5857253

The spectrum of skin biopsies and excisions in a pediatric skin center.

Science.gov (United States)

Theiler, Martin; Neuhaus, Kathrin; Kerl, Katrin; Weibel, Lisa

2017-12-01

Little is known about the spectrum of pediatric skin disorders requiring biopsy/excision, their indication, impact on further management, and the accuracy of clinical diagnosis. We aimed to address these questions in the patient population seen at our Swiss University referral center for Pediatric Dermatology and Plastic Surgery. All skin biopsies/excisions performed in patients aged ≤ 16 years over a period of 2 years were retrospectively analyzed. A total of 506 samples were included. The majority of biopsies/excisions (n = 413, 82%) was performed for tumors, cysts, and hamartomas and 18% for other skin conditions. Malignant tumors were found in 12 samples (2%) from four patients. In 121 (24%) patients, the histopathology had an important impact on patient management. In 80 (16%) cases, the pathology did not match with the clinical diagnosis. In 382 (75%) cases, excision was the treatment of choice. Of these, the indication for surgery was based on patient's request in 181 (47%) cases. Surgical interventions for pediatric skin disorders are performed for diagnostic and therapeutic reasons. In this cohort, histopathology was essential for treatment in one quarter of cases. Skin tumors, cysts, and hamartomas often require excision during childhood, with families' request and esthetic considerations playing an important role. What is Known: • The spectrum of pediatric skin conditions has been studied in outpatient, inpatient, and emergency settings. • In contrast, no data exist on the spectrum of pediatric skin disorders undergoing biopsy/excision specifically. What is New: • We analyze biopsies/excisions in children, focusing on diagnosis, indication, and impact on patient management. • Surgical interventions for skin disorders in children are often performed for tumors and hamartomas with esthetic considerations playing a relevant role. If used for diagnostic purposes, they are often performed to confirm or rule out severe skin disease.

The effect of excise tax increases on cigarette prices in South Africa.

Science.gov (United States)

Linegar, Daniel J; van Walbeek, Corne

2018-01-01

The effectiveness of excise tax increases as a tool for reducing tobacco consumption depends largely on how the tax increases impact the retail price. We estimate this relationship in South Africa for 2001-2015. Statistics South Africa provided disaggregated cigarette price data, used in the calculation of the Consumers' Price Index. Data on the excise tax per cigarette were obtained from Budget Reviews prepared by the National Treasury of South Africa. Regression equations were estimated for each month. The month-on-month change in cigarette prices in February through April was regressed against March's excise tax change to estimate the pass-through coefficient. For the other 9 months, the month-on-month change in cigarette price was regressed against monthly dummy variables to determine the size of the non-tax-related price increase in each of these months. The analysis was performed in both nominal and real (inflation-adjusted) terms. Expressed in real terms, the excise tax was undershifted. A R1.00 (one rand) increase in the excise tax is associated with an increase in the retail price of cigarettes of R0.90 in the pre-2010 period, and R0.49 in the post-2010 period. In the pre-2010 period, the tobacco industry increased the retail price of cigarettes in July/August, independent of the excise tax increase. The discretionary July/August price increases largely disappeared after 2010, primarily because the market became more competitive. The degree of excise tax pass-through, and the magnitude of discretionary increases in cigarette prices, is significantly determined by the competitive environment in the cigarette market. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

The financial burden of reexcising incompletely excised soft tissue sarcomas: a cost analysis.

Science.gov (United States)

Alamanda, Vignesh K; Delisca, Gadini O; Mathis, Shannon L; Archer, Kristin R; Ehrenfeld, Jesse M; Miller, Mark W; Homlar, Kelly C; Halpern, Jennifer L; Schwartz, Herbert S; Holt, Ginger E

2013-09-01

Although survival outcomes have been evaluated between those undergoing a planned primary excision and those undergoing a reexcision following an unplanned resection, the financial implications associated with a reexcision have yet to be elucidated. A query for financial data (professional, technical, indirect charges) for soft tissue sarcoma excisions from 2005 to 2008 was performed. A total of 304 patients (200 primary excisions and 104 reexcisions) were identified. Wilcoxon rank sum tests and χ2 or Fisher's exact tests were used to compare differences in demographics and tumor characteristics. Multivariable linear regression analyses were performed with bootstrapping techniques. The average professional charge for a primary excision was $9,694 and $12,896 for a reexcision (pfinancial burden nearly doubles.

Effects of hyperthermia on radiation-induced chromosome breakage and loss in excision repair deficient Drosophila melanogaster

International Nuclear Information System (INIS)

Mittler, S.

1986-01-01

Hyperthermia increased radiosensitivity with respect to γ-ray induced chromosome loss and breakage in all stages of spermatogenesis in the wild type Oregon R strain of Drosophila melanogaster, whereas hyperthermia increased radiosensitivity to a lesser extent in cn mus(2) 201sup(D1), an excision repair mutant with 0 per cent excision capacity and in mus(3) 308sup(D1), a strain with 24 per cent excision capacity. The differences in hyperthermia-induced radiation sensitivity between the excision repair mutants and the wild strain may be due to the hyperthermia affecting the excision repair mechanism, suggesting that one of the possible mechanisms involved in hyperthermia-increased radiosensitivity is an effect on excision repair. (author)

The effect of DNA repair defects on reproductive performance in nucleotide excision repair (NER) mouse models: an epidemiological approach

NARCIS (Netherlands)

Tsai, P.S.; Nielen, M.; Horst, G.T.J. van der; Colenbrander, B.; Heesterbeek, J.A.P.; Fentener van Vlissingen, J.M.

2005-01-01

In this study, we used an epidemiological approach to analyze an animal database of DNA repair deficient mice on reproductive performance in five Nucleotide Excision Repair (NER) mutant mouse models on a C57BL/6 genetic background, namely CSA, CSB, XPA, XPC [models for the human DNA repair disorders

High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs

DEFF Research Database (Denmark)

Duda, Katarzyna; Lonowski, Lindsey A; Kofoed-Nielsen, Michael

2014-01-01

Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing...... higher genome editing rates. For ZFNs, this approach, combined with delivery of donors as single-stranded oligodeoxynucleotides and nucleases as messenger ribonucleic acid, enabled high knockin efficiencies in demanding applications, including biallelic codon conversion frequencies reaching 30......-70% at high transfection efficiencies and ∼2% at low transfection efficiencies, simultaneous homozygous knockin mutation of two genes with ∼1.5% efficiency as well as generation of cell pools with almost complete codon conversion via three consecutive targeting and FACS events. Observed off-target effects...

Relativistic hydrodynamic evolutions with black hole excision

International Nuclear Information System (INIS)

Duez, Matthew D.; Shapiro, Stuart L.; Yo, H.-J.

2004-01-01

We present a numerical code designed to study astrophysical phenomena involving dynamical spacetimes containing black holes in the presence of relativistic hydrodynamic matter. We present evolutions of the collapse of a fluid star from the onset of collapse to the settling of the resulting black hole to a final stationary state. In order to evolve stably after the black hole forms, we excise a region inside the hole before a singularity is encountered. This excision region is introduced after the appearance of an apparent horizon, but while a significant amount of matter remains outside the hole. We test our code by evolving accurately a vacuum Schwarzschild black hole, a relativistic Bondi accretion flow onto a black hole, Oppenheimer-Snyder dust collapse, and the collapse of nonrotating and rotating stars. These systems are tracked reliably for hundreds of M following excision, where M is the mass of the black hole. We perform these tests both in axisymmetry and in full 3+1 dimensions. We then apply our code to study the effect of the stellar spin parameter J/M 2 on the final outcome of gravitational collapse of rapidly rotating n=1 polytropes. We find that a black hole forms only if J/M 2 2 >1, the collapsing star forms a torus which fragments into nonaxisymmetric clumps, capable of generating appreciable 'splash' gravitational radiation

A novel method of selective removal of human DNA improves PCR sensitivity for detection of Salmonella Typhi in blood samples.

Science.gov (United States)

Zhou, Liqing; Pollard, Andrew J

2012-07-27

Enteric fever is a major public health problem, causing an estimated 21million new cases and 216,000 or more deaths every year. Current diagnosis of the disease is inadequate. Blood culture only identifies 45 to 70% of the cases and is time-consuming. Serological tests have very low sensitivity and specificity. Clinical samples obtained for diagnosis of enteric fever in the field generally have blood, so that even PCR-based methods, widely used for detection of other infectious diseases, are not a straightforward option in typhoid diagnosis. We developed a novel method to enrich target bacterial DNA by selective removal of human DNA from blood samples, enhancing the sensitivity of PCR tests. This method offers the possibility of improving PCR assays directly using clinical specimens for diagnosis of this globally important infectious disease. Blood samples were mixed with ox bile for selective lysis of human blood cells and the released human DNA was then digested with addition of bile resistant micrococcal nuclease. The intact Salmonella Typhi bacteria were collected from the specimen by centrifugation and the DNA extracted with QIAamp DNA mini kit. The presence of Salmonella Typhi bacteria in blood samples was detected by PCR with the fliC-d gene of Salmonella Typhi as the target. Micrococcal nuclease retained activity against human blood DNA in the presence of up to 9% ox bile. Background human DNA was dramatically removed from blood samples through the use of ox bile lysis and micrococcal nuclease for removal of mammalian DNA. Consequently target Salmonella Typhi DNA was enriched in DNA preparations and the PCR sensitivity for detection of Salmonella Typhi in spiked blood samples was enhanced by 1,000 fold. Use of a combination of selective ox-bile blood cell lysis and removal of human DNA with micrococcal nuclease significantly improves PCR sensitivity and offers a better option for improved typhoid PCR assays directly using clinical specimens in diagnosis of

Resistance of 3T3 mouse cells to UV light in relation to excision and transfer of dimers to daughter strands

Energy Technology Data Exchange (ETDEWEB)

Menck, C F.M.; Meneghini, R [Sao Paulo Univ. (Brazil). Inst. de Quimica

1982-04-01

Mouse cells (3T3 line) and human fibroblasts are equally sensitive to UV light. At fluences of 2.0-2.5 J/m/sup 2/ mouse cells excise only 20% of the pyrimidine dimers as compared to 80% excised by human fibroblasts. This fluence allows 37% survival in both cases. Hence, mouse cells are more resistant to the same burden of unexcised dimers. The reason for this increased tolerance to dimers does not seem to be due to a recombinational mechanism, as judged by the fact that only ca. 5% of the dimers are transferred from parental to daughter strands. The transfer of dimers was measured by the Micrococcus luteus UV endonuclease assay, irradiating cells at G/sub 1/ to avoid artifacts arising from introduction of dimers in nascent strands. The possibility of other mechanisms being involved in the process of tolerance to DNA lesions is discussed.

CRISPR/Cas9-Mediated Mutagenesis of Human Pluripotent Stem Cells in Defined Xeno-Free E8 Medium.

Science.gov (United States)

Soh, Chew-Li; Huangfu, Danwei

2017-01-01

The recent advent of engineered nucleases including the CRISPR/Cas9 system has greatly facilitated genome manipulation in human pluripotent stem cells (hPSCs). In addition to facilitating hPSC-based disease studies, the application of genome engineering in hPSCs has also opened up new avenues for cell replacement therapy. To improve consistency and reproducibility of hPSC-based studies, and to meet the safety and regulatory requirements for clinical translation, it is necessary to use a defined, xeno-free cell culture system. This chapter describes protocols for CRISPR/Cas9 genome editing in an inducible Cas9 hPSC-based system, using cells cultured in chemically defined, xeno-free E8 Medium on a recombinant human vitronectin substrate. We detail procedures for the design and transfection of CRISPR guide RNAs, colony selection, and the expansion and validation of clonal mutant lines, all within this fully defined culture condition. These methods may be applied to a wide range of genome-engineering applications in hPSCs, including those that utilize different types of site-specific nucleases such as zinc finger nucleases (ZFNs) and TALENs, and form a closer step towards clinical utility of these cells.

Adjuvant irradiation improves the outcome of patients with rectal cancer following local excision

International Nuclear Information System (INIS)

Chakravarti, Arnab; Willett, Christopher G.; Shellito, Paul; Kaufman, Donald; Daley, William J.

1997-01-01

PURPOSE: the long-term outcomes of patients undergoing local excision with or without pelvic irradiation were examined to define the role of adjuvant irradiation following local excision of T1 and T2 rectal cancers. METHODS: From January 1966 to January 1997, 114 patients underwent local excision for rectal cancer. Fifty-nine patients underwent local excision alone, and 55 patients received adjuvant pelvic irradiation (45 Gy to 65.8 Gy)+/-concurrent 5-fluorouracil chemotherapy. Outcome was assessed by treatment, stage, and pathologic features of tumor grade and lymphatic/venous vessel involvement. RESULTS: Of the 114 patients treated by local excision, 90 had T1/T2 tumors. The 5-year actuarial local control (LC) and recurrence-free survival (RFS) of these 90 patients are shown (with numbers of patients at risk at 5 years) in the table below. The 5-year actuarial local control and recurrence-free survival (RFS) for T1/T2 patients treated by local excision alone was 85% and 77%, respectively. The corresponding values for patients undergoing local excision and adjuvant irradiation alone were 88% and 78%, respectively. All 20 patients treated by adjuvant chemoradiation achieved 5-year actuarial local control and had a recurrence-free survival of 88%. The improved outcomes in the adjuvant radiation group become more impressive when the higher T stage distribution of this group is considered. Fifty-five percent of the group receiving adjuvant radiation were stage T2 compared to only 14% of patients treated by local excision alone. Subgroup analysis was performed on those features recognized as poor prognostic factors including poorly differentiated histology and lymphatic vascular invasion. Patients in the local excision alone group with one or more adverse pathologic features had reduced rates of local control compared to those with the absence of such features (38% vs. 91%, respectively, p=0.02). In comparison, the 5 patients with poor prognostic features treated by

Papillary lesions of the breast: To excise or observe?

Science.gov (United States)

Khan, Sidrah; Diaz, Adrian; Archer, Kellie J; Lehman, Rebecca R; Mullins, Tiffany; Cardenosa, Gilda; Bear, Harry D

2018-05-01

Papillary lesions of the breast range from benign to atypical to malignant. Although papillomas without frank cancer are benign, their management remains controversial. When a core needle biopsy of a lesion yields a diagnosis of intraductal papilloma with atypia, excision is generally recommended to rule out a concurrent malignant neoplasm. For intraductal papillomas without atypia, however, recommendations for excision versus observation are variable. The aims of this study are to evaluate the rate of concurrent malignancies for intraductal papilloma diagnosed on core needle biopsy and to assess the long-term risk of developing cancer after the diagnosis of a papillary lesion. This single institution retrospective study analyzed 259 patients that were diagnosed with intraductal papilloma (IDP) by core needle biopsy from 1995 to 2010. Patients were grouped by initial diagnosis into three groups (papilloma without atypia, papilloma with atypia, and papilloma with atypical duct hyperplasia or atypical lobular hyperplasia (ADH/ALH) and followed up for long-term outcomes. After a core needle biopsy showing IDP with atypia or IDP + ADH/ALH, surgical excision yielded a diagnosis of concomitant invasive or ductal in situ cancer in greater that 30% of cases. For intraductal papilloma without atypia, the likelihood of cancer was much lower. Moreover, even with excision, the finding of intraductal papilloma with atypia carries a significant risk of developing cancer long-term, and such patients should be followed carefully and perhaps should be considered for chemoprevention. © 2017 Wiley Periodicals, Inc.

Size and frequency of gaps in newly synthesized DNA of xeroderma pigmentosum human cells irradiated with ultraviolet light

International Nuclear Information System (INIS)

Meneghini, R.; Cordeiro-Stone, M.; Schumacher, R.I.

1981-01-01

Native newly synthesized DNA from human cells (xeroderma pigmentosum type) irradiated with ultraviolet light releases short pieces of DNA (L-DNA) when incubated with the single-strand specific S 1 nuclease. This is not observed in the case of unirradiated cells. Previous experiments had shown that the L-DNA resulted from the action of S 1 nuclease upon gaps, i.e., single-stranded DNA discontinuities in larger pieces of double-stranded DNA. We verified that the duplex L-DNA, that arises from the inter-gap regions upon S 1 nuclease treatment, has a size which approximates the distance between two pyrimidine dimers on the same strand. A method was devised to measure the size of the gaps. These parameters have been considered in the proposition of a model for DNA synthesis on a template containing pyrimidine dimers

Recurred Myofibroblastoma of Breast After Excision: A Case Report

International Nuclear Information System (INIS)

Cha, Yoon Ki; Kim, Ji Young; Jeong, Myeong Ja; Kim, Jae Hyung; Kim, Soung Hee; Kim, Soo Hyun; Jun, Woo Sun; Park, Kyeong Mee; Yang, Keun Ho

2010-01-01

Myofibroblastoma of the breast is a rare benign mesenchymal tumor that is known to occur in middle-aged and elderly men, yet there are some recent reports showing no certain difference for the gender distribution of this malady. Localized mass excision can usually provide a complete cure. To the best of our knowledge, there have been no reports of metastasis or recurrence of this tumor. Here we describe the sonographic findings of a case of recurrent myofibroblastoma after surgical excision for suspected fibroadenomas in both breasts of a 25-year-old woman

A seventh complementation group in excision-deficient xeroderma pigmentosum

International Nuclear Information System (INIS)

Keijzer, W.; Jaspers, N.G.J.; Bootsma, D.; Abrahams, P.J.; Taylor, A.M.R.; Arlett, C.F.; Zelle, B.; Kinmont, P.D.S.

1979-01-01

Cells from a xeroderma pigmentosum patient XP2B1 who has reached 17 years of age with no keratoses or skin tumours constitute a new, 7th complementation group G. These cells exhibit a low residual level of excision repair, 2% of normal after a UV dose of 5 J/m 2 and an impairment of post-replication repair characteristic of excision-defective XPs. They are also sensitive to the lethal effects of UV and defective in host-cell reactivation of UV-irradiated SV40 DNA. (Auth.)

The influence of pH on the excision of UV-photoproducts from HeLa cells

International Nuclear Information System (INIS)

Masek, F.; Hochmann, J.; Duraj, J.

1977-01-01

Excision of pyrimidine dimers with pH decreasing in UV irradiated HeLa cells can be depressed. This depression is independent of the UV dose within the investigated range. The excision capacity of the HeLa repair system from absolute amount of excised dimers is shown. (author)

Determining Factors for Fast Corneal Sensitivity Recovery After Pterygium Excision

OpenAIRE

Julio Morán, Gemma; Campos, Pamela; Pujol Vives, Pere; Munguia, Aitana; Mas Aixalà, Enric

2016-01-01

Purpose: To establish determining factors for fast corneal sensitivity (CS) recovery after pterygium excision. Methods: Thirty-two eyes of 14 males and 18 females with primary nasal pterygium were recruited. Differences in CS (in the 4 quadrants and the center using a Cochet–Bonnet esthesiometer), pterygium corneal area (PCA), tear osmolarity, tear break-up time, Schirmer test, and ocular symptoms were analyzed before and 1 month after lesion excision. The relationship between CS recovery...

Automated 5 ' nuclease PCR assay for identification of Salmonella enterica

DEFF Research Database (Denmark)

Hoorfar, Jeffrey; Ahrens, Peter; Rådström, P.

2000-01-01

-point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [Delta Rn], >0.6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (Delta Rn, less than or equal to 0.5). All 100 rough...... Salmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at -20 degrees C, Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method......A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica...

[Efficacy evaluation of laparoscopic complete mesocolic excision for transverse colon cancer].

Science.gov (United States)

Cao, Jinpeng; Ji, Yong; Peng, Xiang; Wu, Wenhui; Cheng, Longqing; Zhou, Yonghui; Yang, Ping

2017-05-25

To investigate the safety, feasibility and long-term outcomes of laparoscopic complete mesocolic excision for the transverse colon cancer. Clinical data of 61 patients who underwent laparoscopic complete mesocolic excision for transverse colon cancer (transverse group) in our department from January 2011 to January 2014 were retrospectively analyzed, which were compared with those of 155 patients undergoing laparoscopic complete mesocolic excision for ascending colon cancer (ascending group) and 230 patients undergoing laparoscopic complete mesocolic excision for sigmoid colon cancer (sigmoid group). Differences in operative details, postoperative recovery, postoperative complications and long-term survival among 3 groups were evaluated. No significant differences in the baseline information were found among 3 groups(all P>0.05). The average operative time was significantly longer in transverse group as compared to ascending group and sigmoid group [(192.1±58.7) min vs. (172.2±54.7) min and (169.1±53.6) min]( P0.05). A total of 436 patients received postoperative follow-up of median 36 (5 to 67) months. The overall 5-year survival rate was 73.1%, 73.7% and 74.8%, and the 5-year disease-free survival rate was 71.5%, 71.1% and 72.7% in transverse, ascending and sigmoid colon cancer groups respectively, whose differences were not significant among 3 groups (all P>0.05). Laparoscopic complete mesocolic excision for transverse colon cancer is safe and feasible with slightly longer operation time, and has quite good long-term oncologic efficacy.

Application of halophilic nuclease H of Micrococcus varians subsp. halophilus to commercial production of flavoring agent 5'-GMP.

Science.gov (United States)

Kamekura, M; Hamakawa, T; Onishi, H

1982-01-01

RNA was degraded at 60 degrees C for 24 h by halophilic nuclease H in supernatants from broth cultures of Micrococcus varians subsp. halophilus containing 12% NaCl. Since contaminating 5'-nucleotidase exhibited almost no activity under these conditions, the 5'-GMP formed could be recovered from the reaction mixture, and the yield was 805 mg from 5 g of RNA. PMID:6184020

Connective tissue: cancer patients’ attitudes towards medical research using excised (tumour) tissue

NARCIS (Netherlands)

Vermeulen, E.; Schmidt, M.K.; Cornel, M.C.; Knoppers, B.M.; van Leeuwen, F.E.; Aaronson, N.K.

2011-01-01

The objective of this article is to explore the views of Dutch cancer patients on the use of excised and stored (tumor) tissues in medical research. Excised tissues are routinely stored in hospitals for future diagnostic use. They are also important for scientific research. This article discusses

Investigation of a redox-sensitive predictive model of mouse embryonic stem cells differentiation using quantitative nuclease protection assays and glutathione redox status

Science.gov (United States)

Investigation of a redox-sensitive predictive model of mouse embryonic stem cell differentiation via quantitative nuclease protection assays and glutathione redox status Chandler KJ,Hansen JM, Knudsen T,and Hunter ES 1. U.S. Environmental Protection Agency, Research Triangl...

Pisiform excision for pisotriquetral instability and arthritis.

Science.gov (United States)

Campion, Heather; Goad, Andrea; Rayan, Ghazi; Porembski, Margaret

2014-07-01

To evaluate wrist strength and kinematics after pisiform excision and preservation of its soft tissue confluence for pisotriquetral instability and arthritis. We evaluated 12 patients, (14 wrists) subjectively and objectively an average of 7.5 years after pisiform excision. Three additional patients were interviewed by phone. Subjective evaluation included inquiry about pain and satisfaction with the treatment. Objective testing included measuring wrist flexion and extension range of motion, grip strength, and static and dynamic flexion and ulnar deviation strengths of the operative hand compared with the nonsurgical normal hand. Four patients had concomitant ulnar nerve decompression at the wrist. All patients were satisfied with the outcome. Wrist flexion averaged 99% and wrist extension averaged 95% of the nonsurgical hand. Mean grip strength of the operative hand was 90% of the nonsurgical hand. Mean static flexion strength of the operative hand was 94% of the nonsurgical hand, whereas mean dynamic flexion strength was 113%. Mean static ulnar deviation strength of the operative hand was 87% of the nonsurgical hand. The mean dynamic ulnar deviation strength of the operative hand was 103% of the nonsurgical hand. Soft tissue confluence-preserving pisiform excision relieved pain and retained wrist motion and static and dynamic strength. Associated ulnar nerve compression was a confounding factor that may have affected outcomes. Therapeutic IV. Copyright © 2014 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.

Minimally invasive endoscope-assisted trans-oral excision of huge parapharyngeal space tumors.

Science.gov (United States)

Li, Shang-Yi; Hsu, Ching-Hui; Chen, Mu-Kuan

2015-04-01

Parapharyngeal space tumors are rare head and neck neoplasms, and most are benign lesions. Complete excision of these tumors is difficult because of the complexity of the surrounding anatomic structures. The algorithm for excision of these tumors is typically based on the tumor's characteristics; excision is performed via approaches such as the trans-oral route, the trans-cervical route, and even a combination of the trans-parotid route and mandibulotomy. However, each of these approaches is associated with some complications. Endoscope-assisted minimally invasive surgery is being increasingly employed for surgeries in the head and neck regions. It has the advantage of leaving no facial scars, and ensures better patient comfort after the operation. Here, we report the use of endoscope-assisted trans-oral surgery for excision of parapharyngeal space tumors. The technique yields an excellent outcome and should be a feasible, safe, and economic method for these patients. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Incomplete excision repair process after UV-irradiation in MUT-mutants of Proteus mirabillis

International Nuclear Information System (INIS)

Stoerl, K.

1977-01-01

MUT-mutants of P. mirabilis seem to be able to perform the incision step in the course of excision repair. In contrast to the corresponding wildtype strains with MUT-mutants the number of single-strand breaks formed after UV-irradiation is independent of the UV-dose up to about 720 erg/mm 2 . Incubation in minimal medium over a longer time does not result in completion of excision repair; about 3-6 single-strand breaks in the DNA of these mutants remain open. Likewise, the low molecular weight of the newly synthesized daughter DNA confirms an incompletely proceeding or delayed repair process. As a possible reason for the mutator phenotype an alteration of the DNA-polymerase playing a role in excision and resynthesis steps of excision repair is discussed. (author)

Effects of nucleotide pool imbalances on the excision repair of ultraviolet-induced damage in the DNA of human diploid fibroblasts

International Nuclear Information System (INIS)

Snyder, R.D.

1985-01-01

In an attempt to better understand the mechanism of repair inhibition by DNA polymerase inhibitors, and the nature of hydroxyurea enhancement, experiments were initiated in which the effects of a series of ribonucleotide reductase inhibitors on dNTP pools and on the DNA repair process were determined in both quiescent cultures and log-phase cultures of human fibroblasts. It was determined that hydroxyurea, deoxyadenosine, pyridine-2-carboxaldehyde thiosemicarbazone (TSC), pyrozoloimidazole (IMPY), 3,5-diamino-1,2,4-triazole (guanazole), 3,4,5-trihydroxy benzohydroxamic acid (THBA) and 3,4-dihydroxy benzohydroxamic acid (DHBA) are all effective inhibitors of the DNA repair process in confluent cells but not in log-phase cells. Moreover, the effects of these inhibitors can be reversed by the addition of certain combinations of deoxynucleosides. These reversal studies and the direct analysis of dNTP pool modulation by these compounds in log phase and confluent cultures support the notion that specific pool depletions rather than general imbalance of pools gives rise to the inhibition of the DNA excision repair process

Nuclease-mediated double-strand break (DSB) enhancement of small fragment homologous recombination (SFHR) gene modification in human-induced pluripotent stem cells (hiPSCs).

Science.gov (United States)

Sargent, R Geoffrey; Suzuki, Shingo; Gruenert, Dieter C

2014-01-01

Recent developments in methods to specifically modify genomic DNA using sequence-specific endonucleases and donor DNA have opened the door to a new therapeutic paradigm for cell and gene therapy of inherited diseases. Sequence-specific endonucleases, in particular transcription activator-like (TAL) effector nucleases (TALENs), have been coupled with polynucleotide small/short DNA fragments (SDFs) to correct the most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, a 3-base-pair deletion at codon 508 (delF508), in induced pluripotent stem (iPS) cells. The studies presented here describe the generation of candidate TALENs and their co-transfection with wild-type (wt) CFTR-SDFs into CF-iPS cells homozygous for the delF508 mutation. Using an allele-specific PCR (AS-PCR)-based cyclic enrichment protocol, clonal populations of corrected CF-iPS cells were isolated and expanded.

Base excision repair, aging and health span

Czech Academy of Sciences Publication Activity Database

Xu, G.; Herzig, M.; Rotrekl, Vladimír; Walter, Ch. A.

2008-01-01

Roč. 129, 7-8 (2008), s. 366-382 ISSN 0047-6374 Institutional research plan: CEZ:AV0Z50390512 Keywords : base excision repair * aging * DNA damage Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.915, year: 2008

76 FR 3502 - Time for Payment of Certain Excise Taxes, and Quarterly Excise Tax Payments for Small Alcohol...

Science.gov (United States)

2011-01-20

..., Surety bonds, Virgin Islands, Warehouses. 27 CFR Part 40 Cigars and cigarettes, Claims, Electronic fund... required to pay taxes through electronic funds transfer (EFT), this first payment period ends on September... Part 19 Caribbean Basin Initiative, Claims, Electronic funds transfers, Excise taxes, Exports, Gasohol...

The Relationship between Tax Rate, Penalty Rate, Tax Fairness and Excise Duty Non-compliance.

Directory of Open Access Journals (Sweden)

Sinnasamy Perabavathi

2017-01-01

Full Text Available The rise of indirect tax non-compliance by taxpayers became the main concern of most of the tax authorities around the globe. In Malaysia, non complaince such as smuggling and illegal trade activities by importers involving cigarettes, liquor and imported vehicles bound under Excise Act 1976 have caused revenue losses in monetary and non-monetary aspects. Therefore, the objective of this study is to examine the relationship of tax rate, penalty rate and tax fairness of excise duty non-compliance. This study uses the Deterrence Theory as a basis theory to investigate the phenomenon of excise duty non complaince. A total of 500 excise duty offenders throughout Malaysia responded to the survey. The model was empirically tested by using Partial Least Squares (PLS with disproportionate stratified random sampling technique. The results indicated that the perception of tax rate and penalty rate are positively related while tax fairness is negatively related to excise duty non-compliance among importers.

Results of surgical excision of urethral prolapse in symptomatic patients.

Science.gov (United States)

Hall, Mary E; Oyesanya, Tola; Cameron, Anne P

2017-11-01

Here, we present the clinical presentation and surgical outcomes of women with symptomatic urethral prolapse presenting to our institution over 20 years, and seek to provide treatment recommendations for management of symptomatic urethral prolapse and caruncle. A retrospective review of medical records from female patients who underwent surgery for symptomatic urethral prolapse from June 1995 to August 2015 was performed. Surgical technique consisted of a four-quadrant excisional approach for repair of urethral prolapse. A total of 26 patients were identified with a mean age of 38.8 years (range 3-81). The most common presentations were vaginal bleeding, hematuria, pain, and dysuria. All patients underwent surgical excision of urethral prolapse via a standard approach. Follow-up data was available in 24 patients. Six patients experienced temporary postoperative bleeding, and one patient required placement of a Foley catheter for tamponade. One patient experienced temporary postoperative urinary retention requiring Foley catheter placement. Three patients had visible recurrence of urethral prolapse, for which one later underwent re-excision. Surgical excision of urethral prolapse is a reasonable treatment option in patients who have tried conservative management without relief, as well as in those who present with severe symptoms. Possible complications following excision include postoperative bleeding and recurrence, and patients must be counseled accordingly. In this work, we propose a treatment algorithm for symptomatic urethral prolapse. © 2017 Wiley Periodicals, Inc.

Microendoscopic excision of C2 osteoid osteoma: a technical report.

Science.gov (United States)

Kulkarni, Arvind G; Dhruv, Abhilash N; Bassi, Anupreet J

2013-09-01

Case report and description of technique. To describe a microendoscopic posterior approach for excision of an osteoid osteoma of C2. Microendoscopic techniques are widely used in the management of degenerative disorders of the spine. This is the first report of their use in the management of an osteoid osteoma via the posterior approach. A 12-year-old-boy presented with left-sided neck pain of 3-month duration. Investigations revealed an osteoid osteoma of C2 lamina-lateral mass complex. The patient underwent a posterior microendoscopic excision using 18-mm diameter METRx system (Medtronic Sofamor Danek, Memphis, TN) of tubular retractors. A postoperative computed tomographic scan was done and preoperative and postoperative visual analogue scale and Neck Disability Index were evaluated. The patient was periodically followed up for 1 year. The postoperative computed tomographic scan revealed complete excision of the tumor. The visual analogue scale score for neck pain improved from 3/5 (preoperative) to 0/5 (postoperative) and Neck Disability Index from 33.33 (preoperative) to 0 (postoperative) at 1-year follow-up. Microendoscopic techniques can be extended to excise lesions of the spine. It is a safe procedure in experienced hands. The advantages are minimal morbidity, minimal postoperative pain and discomfort, less analgesic dependence, and better cosmesis. The authors recommend this technique for accessible lesions involving the spine.

TT2014 meeting report on the 12th Transgenic Technology meeting in Edinburgh: new era of transgenic technologies with programmable nucleases in the foreground

Czech Academy of Sciences Publication Activity Database

Beck, Inken; Sedláček, Radislav

2015-01-01

Roč. 24, č. 1 (2015), s. 179-183 ISSN 0962-8819 Institutional support: RVO:68378050 Keywords : Transgenic * Nuclease * Gene Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.054, year: 2015

Three surgical planes identified in laparoscopic complete mesocolic excision for right-sided colon cancer.

Science.gov (United States)

Zhu, Da-Jian; Chen, Xiao-Wu; OuYang, Man-Zhao; Lu, Yan

2016-01-12

Complete mesocolic excision provides a correct anatomical plane for colon cancer surgery. However, manifestation of the surgical plane during laparoscopic complete mesocolic excision versus in computed tomography images remains to be examined. Patients who underwent laparoscopic complete mesocolic excision for right-sided colon cancer underwent an abdominal computed tomography scan. The spatial relationship of the intraoperative surgical planes were examined, and then computed tomography reconstruction methods were applied. The resulting images were analyzed. In 44 right-sided colon cancer patients, the surgical plane for laparoscopic complete mesocolic excision was found to be composed of three surgical planes that were identified by computed tomography imaging with cross-sectional multiplanar reconstruction, maximum intensity projection, and volume reconstruction. For the operations performed, the mean bleeding volume was 73±32.3 ml and the mean number of harvested lymph nodes was 22±9.7. The follow-up period ranged from 6-40 months (mean 21.2), and only two patients had distant metastases. The laparoscopic complete mesocolic excision surgical plane for right-sided colon cancer is composed of three surgical planes. When these surgical planes were identified, laparoscopic complete mesocolic excision was a safe and effective procedure for the resection of colon cancer.

Surgical Excision of Benign Papillomas Diagnosed with Core Biopsy: A Community Hospital Approach

International Nuclear Information System (INIS)

Rozentsvayg, Eka; Carver, Kristen; Borkar, Sunita; Mathew, Melvy; Enis, Sean; Friedman, Paul

2011-01-01

Our goal was to assess the value of surgical excision of benign papillomas of the breast diagnosed on percutaneous core biopsy by determining the frequency of upgrade to malignancies and high risk lesions on a final surgical pathology. We reviewed 67 patients who had biopsies yielding benign papilloma and underwent subsequent surgical excision. Surgical pathology of the excised lesions was compared with initial core biopsy pathology results. 54 patients had concordant benign core and excisional pathology. Cancer (ductal carcinoma in situ and invasive ductal carcinoma) was diagnosed in five (7%) patients. Surgery revealed high-risk lesions in 8 (12%) patients, including atypical ductal hyperplasia, atypical lobular hyperplasia, and lobular carcinoma in situ. Cancer and high risk lesions accounted for 13 (19%) upstaging events from benign papilloma diagnosis. Our data suggests that surgical excision is warranted with core pathology of benign papilloma

Plant multifunctional nuclease TBN1 with unexpected phospholipase activity: structural study and reaction-mechanism analysis

Czech Academy of Sciences Publication Activity Database

Koval, Tomáš; Lipovová, P.; Podzimek, T.; Matoušek, Jaroslav; Dušková, Jarmila; Skálová, Tereza; Štěpánková, Andrea; Hašek, Jindřich; Dohnálek, Jan

2013-01-01

Roč. 69, č. 2 (2013), s. 213-226 ISSN 0907-4449 R&D Projects: GA MŠk EE2.3.30.0029; GA ČR GAP302/11/0855; GA ČR GA310/09/1407; GA ČR GA521/09/1214 Grant - others:AV ČR(CZ) AP0802 Program:Akademická prémie - Praemium Academiae Institutional support: RVO:61389013 ; RVO:60077344 Keywords : plant nucleases * catalytic zinc cluster * glycoproteins Subject RIV: CE - Biochemistry; EB - Genetics ; Molecular Biology (BC-A) Impact factor: 7.232, year: 2013

Robotic transanal total mesorectal excision for rectal cancer: experience with a first case

NARCIS (Netherlands)

Verheijen, P.M.; Consten, E.C.J.; Broeders, Ivo Adriaan Maria Johannes

2014-01-01

Background: A transanal approach for total mesorectal excision (TME) using a single incision port is feasible. The disadvantages are technical difficulties associated with limited manoeuvrability. Methods: We present our first experience with robotic-assisted transanal total mesorectal excision. A

R-loops: targets for nuclease cleavage and repeat instability.

Science.gov (United States)

Freudenreich, Catherine H

2018-01-11

R-loops form when transcribed RNA remains bound to its DNA template to form a stable RNA:DNA hybrid. Stable R-loops form when the RNA is purine-rich, and are further stabilized by DNA secondary structures on the non-template strand. Interestingly, many expandable and disease-causing repeat sequences form stable R-loops, and R-loops can contribute to repeat instability. Repeat expansions are responsible for multiple neurodegenerative diseases, including Huntington's disease, myotonic dystrophy, and several types of ataxias. Recently, it was found that R-loops at an expanded CAG/CTG repeat tract cause DNA breaks as well as repeat instability (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Two factors were identified as causing R-loop-dependent breaks at CAG/CTG tracts: deamination of cytosines and the MutLγ (Mlh1-Mlh3) endonuclease, defining two new mechanisms for how R-loops can generate DNA breaks (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Following R-loop-dependent nicking, base excision repair resulted in repeat instability. These results have implications for human repeat expansion diseases and provide a paradigm for how RNA:DNA hybrids can cause genome instability at structure-forming DNA sequences. This perspective summarizes mechanisms of R-loop-induced fragility at G-rich repeats and new links between DNA breaks and repeat instability.

Ionic charge transport between blockages: Sodium cation conduction in freshly excised bulk brain tissue

Energy Technology Data Exchange (ETDEWEB)

Emin, David, E-mail: emin@unm.edu [Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM 87131 (United States); Akhtari, Massoud [Semple Institutes for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095 (United States); Ellingson, B. M. [Department of Radiology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095 (United States); Mathern, G. W. [Department of Neurosurgery, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095 (United States)

2015-08-15

We analyze the transient-dc and frequency-dependent electrical conductivities between blocking electrodes. We extend this analysis to measurements of ions’ transport in freshly excised bulk samples of human brain tissue whose complex cellular structure produces blockages. The associated ionic charge-carrier density and diffusivity are consistent with local values for sodium cations determined non-invasively in brain tissue by MRI (NMR) and diffusion-MRI (spin-echo NMR). The characteristic separation between blockages, about 450 microns, is very much shorter than that found for sodium-doped gel proxies for brain tissue, >1 cm.

76 FR 3584 - Time for Payment of Certain Excise Taxes, and Quarterly Excise Tax Payments for Small Alcohol...

Science.gov (United States)

2011-01-20

... 40 Cigars and cigarettes, Claims, Electronic fund transfers, Excise taxes, Labeling, Packaging and..., Regulations.gov , we will post, and you may view, copies of this notice, any electronic or mailed comments we... material that we consider unsuitable for posting. You also may view copies of this notice, any electronic...

Pathogenicity Island Cross Talk Mediated by Recombination Directionality Factors Facilitates Excision from the Chromosome.

Science.gov (United States)

Carpenter, Megan R; Rozovsky, Sharon; Boyd, E Fidelma

2015-12-14

Pathogenicity islands (PAIs) are mobile integrated genetic elements (MIGEs) that contain a diverse range of virulence factors and are essential in the evolution of pathogenic bacteria. PAIs are widespread among bacteria and integrate into the host genome, commonly at a tRNA locus, via integrase-mediated site-specific recombination. The excision of PAIs is the first step in the horizontal transfer of these elements and is not well understood. In this study, we examined the role of recombination directionality factors (RDFs) and their relationship with integrases in the excision of two PAIs essential for Vibrio cholerae host colonization: Vibrio pathogenicity island 1 (VPI-1) and VPI-2. VPI-1 does not contain an RDF, which allowed us to answer the question of whether RDFs are an absolute requirement for excision. We found that an RDF was required for efficient excision of VPI-2 but not VPI-1 and that RDFs can induce excision of both islands. Expression data revealed that the RDFs act as transcriptional repressors to both VPI-1- and VPI-2-encoded integrases. We demonstrated that the RDFs Vibrio excision factor A (VefA) and VefB bind at the attachment sites (overlapping the int promoter region) of VPI-1 and VPI-2, thus supporting this mode of integrase repression. In addition, V. cholerae RDFs are promiscuous due to their dual functions of promoting excision of both VPI-1 and VPI-2 and acting as negative transcriptional regulators of the integrases. This is the first demonstration of cross talk between PAIs mediated via RDFs which reveals the complex interactions that occur between separately acquired MIGEs. Deciphering the mechanisms of pathogenicity island excision is necessary for understanding the evolution and spread of these elements to their nonpathogenic counterparts. Such mechanistic insight would assist in predicting the mobility of uncharacterized genetic elements. This study identified extensive RDF-mediated cross talk between two nonhomologous VPIs and

Cigarette Excise Taxes in Context: Cautionary Lessons from the U.S. Experience.

Science.gov (United States)

Campbell, Richard B; Balbach, Edith D

2015-01-01

Cigarette excise taxes are an important tool in the World Health Organization Framework Convention on Tobacco Control strategy for reducing global tobacco consumption. However, contemporary tobacco control efforts also coincide with the proliferation of neoliberal economic programs calling for the withdrawal of state activity from the economy to facilitate trade. In this environment, cigarette excise taxes may be seen less as an instrument of tobacco control than a feature of an economic program that is punitive to lower-income people. This article reviews collaboration between progressive organizations in the United States and the tobacco industry in the 1980s and 1990s, documenting potential sources of unanticipated resistance to excise taxes and highlighting the tobacco industry's capacity to engage in policy issues through third-party surrogates. It is important for those implementing cigarette excise tax increases to distance tobacco control objectives from larger economic policy measures and for tobacco control advocates to build alliances with organizations working for economic fairness in order to address mutual concerns. © SAGE Publications 2015.

A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering.

Science.gov (United States)

Nørholm, Morten H H

2010-03-16

The combined use of restriction enzymes with PCR has revolutionized molecular cloning, but is inherently restricted by the content of the manipulated DNA sequences. Uracil-excision based cloning is ligase and sequence independent and allows seamless fusion of multiple DNA sequences in simple one-tube reactions, with higher accuracy than overlapping PCR. Here, the addition of a highly efficient DNA polymerase and a low-background-, large-insertion- compatible site-directed mutagenesis protocol is described, largely expanding the versatility of uracil-excision DNA engineering. The different uracil-excision based molecular tools that have been developed in an open-source fashion, constitute a comprehensive, yet simple and inexpensive toolkit for any need in molecular cloning.

Union women, the tobacco industry, and excise taxes: a lesson in unintended consequences.

Science.gov (United States)

Balbach, Edith D; Campbell, Richard B

2009-08-01

Between 1987 and 1997, the tobacco industry used the issue of cigarette excise tax increases to create a political partnership with the Coalition of Labor Union Women (CLUW), a group representing female trade unionists in the U.S. This paper documents how the industry created this relationship and the lessons tobacco-control advocates can learn from the industry's example, in order to mitigate possible unintended consequences of advocating excise tax increases. In 1998, under the terms of the Master Settlement Agreement, the tobacco industry began making documents produced in litigation available publicly. Currently, approximately 50 million pages are available online, including substantial documentation of the industry-CLUW relationship. For this study, a comprehensive search of these documents was conducted. The tobacco industry encouraged CLUW's opposition to excise tax increases by emphasizing the economic regressivity of these taxes, discussing excise taxes generically to deflect attention from cigarettes, and encouraging opposition to earmarking cigarette taxes to pay for specific programs. In addition, CLUW received at least $221,500 in financial support between 1987 and 1997 and in-kind support for its conferences, membership materials, and other services. Excise tax increases, if pursued without considering the impacts they may have on low-SES populations, may have unintended consequences. In this case, such proposals may have helped to create a relationship between CLUW and the tobacco industry. Because excise taxes are endorsed in the Framework Convention on Tobacco Control, tobacco-control advocates must understand how to build relationships with low-SES populations and mitigate potential alliances with the tobacco industry.

The Impact of Pterygium Excision on Corneal Astigmatism

International Nuclear Information System (INIS)

Khan, F. A.; Niazi, S. P. K.; Khan, D. A.

2014-01-01

Objective: To compare the corneal astigmatism before and after the excision of pterygium and also to determine the correlation of pterygium size with the postoperative corneal astigmatism. Study Design: Cross-sectional interventional study. Place and Duration of Study: Eye Department, Combined Military Hospital, Abbottabad, from May 2011 to March 2012. Methodology: Thirty patients underwent pterygium excision. Pre-operatively Snellen visual acuity, manifest refraction and slit lamp examination was done. The size of the pterygium was recorded in mm by projecting a horizontal slit lamp beam from the limbus to the apex. All the pterygium were equal to or greater than 2.5 mm. Keratometry was performed with an automated keratometer. Keratometric data was recorded pre-operatively and at 28 days postoperatively. Wilcoxon signed rank test was used for comparing the pre-operative and the postoperative corneal astigmatism. Spearman's rank order was calculated to observe correlation of pterygium size with the postoperative astigmatism. Results: The median (mean rank) pre-operative astigmatism of 2.25 (15.50) reduced to a median (mean rank) postoperative astigmatism of 1.30 (14.96). This decrease in the postoperative astigmatism was statistically significant (p < 0.001). There was a statistically non-significant correlation between the postoperative astigmatism and the pterygium size (rs = -0.29, p = 0.12). Conclusion: Pterygium excision caused significant reduction in corneal astigmatism. (author)

Oesophageal pseudodiverticulum after foregut duplication cyst excision: Case report and literature review

Directory of Open Access Journals (Sweden)

Iuliana D Bobanga

2016-01-01

Full Text Available Oesophageal pseudodiverticula rarely occur after excision of benign oesophageal neoplasms. While management and outcomes have been reported in the adult leiomyoma literature, sparse data exist on the occurrence and management of pseudodiverticula after foregut duplication cyst excision. We discuss our experience with a paediatric patient and review relevant literature regarding operative techniques and surgical outcomes.

Structural and Functional Studies on Nucleotide Excision Repair From Recognition to Incision.

Energy Technology Data Exchange (ETDEWEB)

Caroline Kisker

2001-01-01

Maintenance of the correct genetic information is crucial for all living organisms because mutations are the primary cause of hereditary diseases, as well as cancer and may also be involved in aging. The importance of genomic integrity is underscored by the fact that 80 to 90% of all human cancers are ultimately due to DNA damage. Among the different repair mechanisms that have evolved to protect the genome, nucleotide excision repair (NER) is a universal pathway found in all organisms. NER removes a wide variety of bulky DNA adducts including the carcinogenic cyclobutane pyrimidine dimers induced by UV radiation, benzo(a)pyrene-guanine adducts caused by smoking and the guanine-cisplatin adducts induced by chemotherapy. The importance of this repair mechanism is reflected by three severe inherited diseases in humans, which are due to defects in NER: xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy.

CRISPR-Cas9 System as a Versatile Tool for Genome Engineering in Human Cells

Directory of Open Access Journals (Sweden)

Xuelian Wang

2016-01-01

Full Text Available Targeted nucleases are influential instruments for intervening in genome revision with great accuracy. RNA-guided Cas9 nucleases produced from clustered regularly interspaced short palindromic repeats (CRISPR-Cas systems have noticeably altered the means to modify the genomes of distinct organisms. They can be notably used to facilitate effective genome manipulation in eukaryotic cells by clearly detailing a 20-nt targeting sequence inside its directed RNA. We discuss the most recent advancements in the molecular basis of the type II CRISPR/Cas system and encapsulate applications and elements affecting its use in human cells. We also propose possible applications covering its uses ranging from basic science to implementation in the clinic.

Comparison between preoperative biopsy and post-excision ...

African Journals Online (AJOL)

peripheral nerve sheath tumour (6%).[6] Soft-tissue sarcomas most frequently affect the extremities and include MFH (40%), lipo- sarcoma (25%), synovial sarcoma and fibrosarcoma.[7]. Appropriate management is reliant on an accurate preoperative histology result. Excision biopsy is recommended for tumours. <3 cm in ...

Uracil Excision for Assembly of Complex Pathways

DEFF Research Database (Denmark)

Cavaleiro, Mafalda; Nielsen, Morten Thrane; Kim, Se Hyeuk

2015-01-01

Despite decreasing prices on synthetic DNA constructs, higher-order assembly of PCR-generated DNA continues to be an important exercise in molecular and synthetic biology. Simplicity and robustness are attractive features met by the uracil excision DNA assembly method, which is one of the most in...

Cigarette price minimization strategies in the United States: price reductions and responsiveness to excise taxes.

Science.gov (United States)

Pesko, Michael F; Licht, Andrea S; Kruger, Judy M

2013-11-01

Because cigarette price minimization strategies can provide substantial price reductions for individuals continuing their usual smoking behaviors following federal and state cigarette excise tax increases, we examined independent price reductions compensating for overlapping strategies. The possible availability of larger independent price reduction opportunities in states with higher cigarette excise taxes is explored. Regression analysis used the 2006-2007 Tobacco Use Supplement of the Current Population Survey (N = 26,826) to explore national and state-level independent price reductions that smokers obtained from purchasing cigarettes (a) by the carton, (b) in a state with a lower average after-tax cigarette price than in the state of residence, and (c) in "some other way," including online or in another country. Price reductions from these strategies are estimated jointly to compensate for known overlapping strategies. Each strategy reduced the price of cigarettes by 64-94 cents per pack. These price reductions are 9%-22% lower than conventionally estimated results not compensating for overlapping strategies. Price reductions vary substantially by state. Following cigarette excise tax increases, the price reduction available from purchasing cigarettes by cartons increased. Additionally, the price reduction from purchasing cigarettes in a state with a lower average after-tax cigarette price is positively associated with state cigarette excise tax rates and border state cigarette excise tax rate differentials. Findings from this large, nationally representative study of cigarette smokers suggest that price reductions are larger in states with higher cigarette excise taxes, and increase as cigarette excise taxes rise.

THE IMPACT OF VISIONARY LEADERSHIP, LEARNING ORGANIZATION AND INNOVATIVE BEHAVIOR TO PERFORMANCE OF CUSTOMS AND EXCISE FUNCTIONAL

OpenAIRE

Anshar, Muhammad

2017-01-01

This research aims to determine the impact of visionary leadership, learning organization, and innovative behavior on the performance of Functional Officers of Customs and Excise Inspectors at Tanjung Priok Customs and Excise Main Service Office. The research was conducted at Tanjung Priok Customs and Excise Service Office using the number of samples of 78 Functional Officers of Customs and Excise Inspectors. The sample was selected using simple random sampling technique. Data collection was ...

Outcome of excision of megarectum in children with anorectal malformation.

Science.gov (United States)

Keshtgar, Alireza S; Ward, Harry C; Richards, Catherine; Clayden, Graham S

2007-01-01

Megarectum in association with anorectal malformation contributes to chronic constipation and fecal incontinence. Resection of megarectum in anorectal malformation improves bowel function, but neuropathy and poor sphincter quality may affect the outcome of fecal continence adversely. The aim of this study was to evaluate the benefits of resection of megarectum in anorectal malformation and to ascertain the impact of anal sphincter quality and neuropathy on the outcome. We studied 62 children with intractable fecal incontinence after repair of anorectal malformation between January 1991 and January 2005. All patients were investigated with anorectal manometry and anal endosonography under ketamine anesthesia. On endosonography, an intact or scarred internal anal sphincter (IAS) was classified as good and a fragmented or absent IAS as poor. On manometry, a resting anal sphincter pressure equal to or more than 30 mm Hg was classified as good and a lower pressure as poor. Functional assessment of fecal continence was done before and after excision of megarectum using a modified Wingfield scores. Sixteen children had excision of megarectum with median age of 9 years (range, 2-15 years) and postoperative follow-up of 5 years (range, 1-10 years). Seven had formation of antegrade continent enema stoma before excision of megarectum. Children were classified into three groups of anomalies: low (n = 6), intermediate (n = 4), and high (n = 6). All children were incontinent of feces. After excision of megarectum, of the 9 children with good IAS and no neuropathy, 7 became continent of feces. Of the remaining 7 children, 4 had poor IAS and 3 had neuropathy, 5 of whom required an antegrade continent enema stoma to be clean. Excision of megarectum in children who had previous repair of anorectal malformation results in fecal continence in the presence of a good IAS and absence of neuropathy. Patients with a poor IAS or neuropathy will often require artificial means of fecal

Knockout of Myostatin by Zinc-finger Nuclease in Sheep Fibroblasts and Embryos

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Xuemei Zhang

2016-10-01

Full Text Available Myostatin (MSTN can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause “double-muscling” trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos.

Orange Peel Excision of Gland: A Novel Surgical Technique for Treatment of Gynecomastia.

Science.gov (United States)

S S, Shirol

2016-12-01

Gynecomastia is a common aesthetic problem faced by men with reported incidence as high as 65% with serious psychosocial impact. Although various techniques of liposculpture combined with glandular excision is the standard of treatment, many of the glandular excision techniques have inherent limitations and complications such as leaving a long scar, long operative time, contour abnormalities, and increased risk of hematoma. Here, we describe an innovative "the orange peel excision of gland (OPEG) technique" which overcomes these limitations with excellent cosmetic results. A total of 38 breasts were operated in 20 patients (18 bilateral and 2 unilateral). All the patients underwent suction-assisted liposuction and glandular excision under general anesthesia by our OPEG technique. The average operative time per breast was 60 minutes. One patient had a small hematoma which did not require evacuation. The patient satisfaction rate was 95%. The technique has reduced operative time and avoids residual gland and hematoma with excellent aesthetic outcome.

Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification.

Science.gov (United States)

Li, Lixin; Piatek, Marek J; Atef, Ahmed; Piatek, Agnieszka; Wibowo, Anjar; Fang, Xiaoyun; Sabir, J S M; Zhu, Jian-Kang; Mahfouz, Magdy M

2012-03-01

Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants.

77 FR 43157 - Disregarded Entities and the Indoor Tanning Services Excise Tax; Correction

Science.gov (United States)

2012-07-24

... Disregarded Entities and the Indoor Tanning Services Excise Tax; Correction AGENCY: Internal Revenue Service... tax. DATES: This correction is effective on July 24, 2012, and applies on and after June 25, 2012. FOR... Subjects in 26 CFR Part 301 Employment taxes, Estate taxes, Excise taxes, Gift taxes, Income taxes...

TREATMENT OF DOGS WITH ORAL MELANOMA RECURRENCE BY DIODE LASER EXCISION

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Cornel Igna

2016-11-01

Full Text Available Introduction: Treatment of oral melanomas utilizes the surgical excision-resection (Culp et al., 2013 and/or radiation therapy (Proulx et al., 2003, chemotherapy with carboplatin (Brockley et al., 2013, immunotherapy (Ottnod et al., 2013. Treatment based on surgical excision is usually palliative (Freeman et al., 2003. Aims: In the literature even though there are data concerning the prognosis of oral melanomas in dogs after surgery, are missing data after laser excision. Taking into account these findings we wished to present our experience regarding three cases of oral melanoma recurrence and immediate and long term laser surgery results. Materials and Methods: The casuistry consisted of three dogs with recurrent oral malignant melanomas, subjected to surgical reintervention. The initial diagnosis was melanotic melanoma in stage I or II. The animals were brought back at different time intervals from originally excision with electric scalpel. Before reintervention, dogs were subjected to clinical, paraclinical exam and biopsy. Excision of the tumor mass was made with an optical fiber hawing a diameter of 400µm, at a power of 10W and a wavelength of 940 nm with a diode laser. At 1, 2, 3, 6 and 12 months after laser reintervention the dogs were reexamined. Results: Average time in which appeared canine oral melanoma relapse was 58.6 days. After reexamination all cases where reinstatement in stage I. Operators times were held in conditions of comfort with wide access, minimum bleeding, effective hemostasis. After surgery at 24 hours on the intervention place a slight local redness, without swelling and bleeding was observed. Palpation revealed initially also a slight local sensitivity which completely disappeared in 48 hours. There were no grasping and chewing disturbances. Macroscopic healing occurred in 7-9 days. At last recheck performed at 12 months there were no evidences of tumour recurrence or metastasis. Conclusion: Diode laser excision

26 CFR 55.4981-2 - Imposition of excise tax with respect to certain undistributed income of real estate investment...

Science.gov (United States)

2010-04-01

... certain undistributed income of real estate investment trusts; calendar years beginning after December 31... (CONTINUED) MISCELLANEOUS EXCISE TAXES (CONTINUED) EXCISE TAX ON REAL ESTATE INVESTMENT TRUSTS AND REGULATED INVESTMENT COMPANIES Excise Tax on Real Estate Investment Trusts § 55.4981-2 Imposition of excise tax with...

Abscisic Acid accumulates at positive turgor potential in excised soybean seedling growing zones.

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Creelman, R A; Mullet, J E

1991-04-01

Abscisic acid (ABA) accumulated in soybean (Glycine max [L.] Merr. cv Williams) hypocotyl elongating regions when seedlings were transferred to low water potential vermiculite (Psi = -0.3 megapascals) even though positive turgor is retained in this tissue. Accumulation of ABA in growing zones could occur from de novo biosynthesis within this tissue or transport from adjacent nongrowing zones. Both growing and nongrowing hypocotyl and root tissues accumulated significant levels of ABA when excised and dehydrated to reduce turgor. Surprisingly, excised growing zones (which experienced no water loss) also accumulated ABA when incubated in darkness for 4 hours at 100% relative humidity and 29 degrees C. Induction of ABA accumulation in the excised elongating region of the hypocotyl was not caused by disruption of root pressure or wounding. While excision of hypocotyl elongating regions induced ABA accumulation, no change in either extensin or p33 mRNA levels was observed. Accumulation of extensin or p33 mRNA required more severe wounding. This suggests that ABA is not involved in the response of these genes in wounded tissue and that wound signals are not causing ABA accumulation in excised tissue. Accumulation of ABA in excised elongating regions was correlated with growth inhibition and a decline in turgor to the yield threshold (Psi;(p) = 0.37 megapascals; R Matyssek, S Maruyama, JS Boyer [1988] Plant Physiol 86: 1163-1167). Inhibiting hypocotyl growth by transferring seedlings to lower temperatures or light did not cause ABA accumulation. We conclude that induction of ABA accumulation in growing zones is more sensitive to changes in turgor than the induction which occurs in mature tissues.

Genomic Knockout of Endogenous Canine P-Glycoprotein in Wild-Type, Human P-Glycoprotein and Human BCRP Transfected MDCKII Cell Lines by Zinc Finger Nucleases.

Science.gov (United States)

Gartzke, Dominik; Delzer, Jürgen; Laplanche, Loic; Uchida, Yasuo; Hoshi, Yutaro; Tachikawa, Masanori; Terasaki, Tetsuya; Sydor, Jens; Fricker, Gert

2015-06-01

To investigate whether it is possible to specifically suppress the expression and function of endogenous canine P-glycoprotein (cPgp) in Madin-Darby canine kidney type II cells (MDCKII) transfected with hPGP and breast cancer resistance protein (hBCRP) by zinc finger nuclease (ZFN) producing sequence specific DNA double strand breaks. Wild-type, hPGP-transfected, and hBCRP-transfected MDCKII cells were transfected with ZFN targeting for cPgp. Net efflux ratios (NER) of Pgp and Bcrp substrates were determined by dividing efflux ratios (basal-to-apical / apical-to-basal) in over-expressing cell monolayers by those in wild-type ones. From ZFN-transfected cells, cell populations (ko-cells) showing knockout of cPgp were selected based on genotyping by PCR. qRT-PCR analysis showed the significant knock-downs of cPgp and interestingly also cMrp2 expressions. Specific knock-downs of protein expression for cPgp were shown by western blotting and quantitative targeted absolute proteomics. Endogenous canine Bcrp proteins were not detected. For PGP-transfected cells, NERs of 5 Pgp substrates in ko-cells were significantly greater than those in parental cells not transfected with ZFN. Similar result was obtained for BCRP-transfected cells with a dual Pgp and Bcrp substrate. Specific efflux mediated by hPGP or hBCRP can be determined with MDCKII cells where cPgp has been knocked out by ZFN.

The Bacillus anthracis chromosome contains four conserved, excision-proficient, putative prophages

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Sozhamannan Shanmuga

2006-04-01

Full Text Available Abstract Background Bacillus anthracis is considered to be a recently emerged clone within the Bacillus cereus sensu lato group. The B. anthracis genome sequence contains four putative lambdoid prophages. We undertook this study in order to understand whether the four prophages are unique to B. anthracis and whether they produce active phages. Results More than 300 geographically and temporally divergent isolates of B. anthracis and its near neighbors were screened by PCR for the presence of specific DNA sequences from each prophage region. Every isolate of B. anthracis screened by PCR was found to produce all four phage-specific amplicons whereas none of the non-B. anthracis isolates, produced more than one phage-specific amplicon. Excision of prophages could be detected by a PCR based assay for attP sites on extra-chromosomal phage circles and for attB sites on phage-excised chromosomes. SYBR-green real-time PCR assays indicated that prophage excision occurs at very low frequencies (2 × 10-5 - 8 × 10-8/cell. Induction with mitomycin C increased the frequency of excision of one of the prophages by approximately 250 fold. All four prophages appear to be defective since, mitomycin C induced culture did not release any viable phage particle or lyse the cells or reveal any phage particle under electron microscopic examination. Conclusion The retention of all four putative prophage regions across all tested strains of B. anthracis is further evidence of the very recent emergence of this lineage and the prophage regions may be useful for differentiating the B. anthracis chromosome from that of its neighbors. All four prophages can excise at low frequencies, but are apparently defective in phage production.

Transaxillary Subcutaneouscopic Excision of Fibroadenoma of the Breast in Children: The Covert Scar Approach.

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Gonzalez, Mirtha; Pimpalwar, Ashwin

2016-02-01

Fibroadenoma is a common condition in adolescent girls. Most tumors are excised either through a periareolar approach or the inframammary approach. Both approaches produce visible scars in adolescent girls. We propose a new cosmetic approach to this lesion and report our experience with the transaxillary subcutaneouscopic approach for excision of the fibroadenoma of the breast. The purpose of this case report is to delineate an innovative surgical approach to resection of a breast fibroadenoma that yields an adequate resection without possible damage to the ductal system while optimizing cosmetic results by avoiding scars. We retrospectively reviewed the medical records of four adolescent girls who underwent the above approach for excision of fibroadenoma of the breast. The age range was 14-16 years. There were no complications in all 4 patients. The final result at the 3-month follow-up revealed an esthetically pleasing skin incision that healed well and was hidden by the natural skin fold of the axilla. Transaxillary subcutaneouscopic excision of fibroadenoma of the breast in children is a safe and effective technique and should be considered for excision of benign breast lesions in children.

Base excision repair activities differ in human lung cancer cells and corresponding normal controls

DEFF Research Database (Denmark)

Karahalil, Bensu; Bohr, Vilhelm A; De Souza-Pinto, Nadja C

2010-01-01

Oxidative damage to DNA is thought to play a role in carcinogenesis by causing mutations, and indeed accumulation of oxidized DNA bases has been observed in samples obtained from tumors but not from surrounding tissue within the same patient. Base excision repair (BER) is the main pathway...... for the repair of oxidized modifications both in nuclear and mitochondrial DNA. In order to ascertain whether diminished BER capacity might account for increased levels of oxidative DNA damage in cancer cells, the activities of BER enzymes in three different lung cancer cell lines and their non......-cancerous counterparts were measured using oligonucleotide substrates with single DNA lesions to assess specific BER enzymes. The activities of four BER enzymes, OGG1, NTH1, UDG and APE1, were compared in mitochondrial and nuclear extracts. For each specific lesion, the repair activities were similar among the three...

TH-AB-209-12: Tissue Equivalent Phantom with Excised Human Tissue for Assessing Clinical Capabilities of Coherent Scatter Imaging Applications

Energy Technology Data Exchange (ETDEWEB)

Albanese, K; Morris, R; Spencer, J [Medical Physics Graduate Program, Duke University, Durham, NC (United States); Greenberg, J [Dept. of Electrical and Computer Engineering, Duke University, Durham, NC (United States); Kapadia, A [Carl E Ravin Advanced Imaging Laboratories, Durham, NC (United States)

2016-06-15

Purpose: Previously we reported the development of anthropomorphic tissue-equivalent scatter phantoms of the human breast. Here we present the first results from the scatter imaging of the tissue equivalent breast phantoms for breast cancer diagnosis. Methods: A breast phantom was designed to assess the capability of coded aperture coherent x-ray scatter imaging to classify different types of breast tissue (adipose, fibroglandular, tumor). The phantom geometry was obtained from a prone breast geometry scanned on a dedicated breast CT system. The phantom was 3D printed using the segmented DICOM breast CT data. The 3D breast phantom was filled with lard (as a surrogate for adipose tissue) and scanned in different geometries alongside excised human breast tissues (obtained from lumpectomy and mastectomy procedures). The raw data were reconstructed using a model-based reconstruction algorithm and yielded the location and form factor (i.e., momentum transfer (q) spectrum) of the materials that were imaged. The measured material form factors were then compared to the ground truth measurements acquired by x-ray diffraction (XRD) imaging. Results: Our scatter imaging system was able to define the location and composition of the various materials and tissues within the phantom. Cancerous breast tissue was detected and classified through automated spectral matching and an 86% correlation threshold. The total scan time for the sample was approximately 10 minutes and approaches workflow times for clinical use in intra-operative or other diagnostic tasks. Conclusion: This work demonstrates the first results from an anthropomorphic tissue equivalent scatter phantom to characterize a coherent scatter imaging system. The functionality of the system shows promise in applications such as intra-operative margin detection or virtual biopsy in the diagnosis of breast cancer. Future work includes using additional patient-derived tissues (e.g., human fat), and modeling additional organs

Aerodynamic and Nonlinear Dynamic Acoustic Analysis of Tension Asymmetry in Excised Canine Larynges

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Devine, Erin E.; Bulleit, Erin E.; Hoffman, Matthew R.; McCulloch, Timothy M.; Jiang, Jack J.

2012-01-01

Purpose: To model tension asymmetry caused by superior laryngeal nerve paralysis (SLNP) in excised larynges and apply perturbation, nonlinear dynamic, and aerodynamic analyses. Method: SLNP was modeled in 8 excised larynges using sutures and weights to mimic cricothyroid (CT) muscle function. Weights were removed from one side to create tension…

THE IMPACT OF VISIONARY LEADERSHIP, LEARNING ORGANIZATION AND INNOVATIVE BEHAVIOR TO PERFORMANCE OF CUSTOMS AND EXCISE FUNCTIONAL

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Muhammad Anshar

2017-12-01

Full Text Available This research aims to determine the impact of visionary leadership, learning organization, and innovative behavior on the performance of Functional Officers of Customs and Excise Inspectors at Tanjung Priok Customs and Excise Main Service Office. The research was conducted at Tanjung Priok Customs and Excise Service Office using the number of samples of 78 Functional Officers of Customs and Excise Inspectors. The sample was selected using simple random sampling technique. Data collection was done by using questionnaire and data analysis using path analysis. The results showed that 1 visionary leadership, learning organization and innovative behavior have a direct and positive impact on performance, 2 visionary leadership, and learning organization have a direct and positive impact on innovative behavior and 3 visionary leadership have a direct and positive impact on learning organization. The conclusion of this research is to improve the performance of Functional Officer of Customs and Excise Inspectors at TanjungPriok Customs and Excise Main Service Office, it is necessary to increase the aspect of visionary leadership from Head of Main Service Office, learning organization at Main Service Office and innovative behavior at Functional Officers of Customs and Excise Inspectors.

Low-Dose Irradiation Enhances Gene Targeting in Human Pluripotent Stem Cells.

Science.gov (United States)

Hatada, Seigo; Subramanian, Aparna; Mandefro, Berhan; Ren, Songyang; Kim, Ho Won; Tang, Jie; Funari, Vincent; Baloh, Robert H; Sareen, Dhruv; Arumugaswami, Vaithilingaraja; Svendsen, Clive N

2015-09-01

Human pluripotent stem cells (hPSCs) are now being used for both disease modeling and cell therapy; however, efficient homologous recombination (HR) is often crucial to develop isogenic control or reporter lines. We showed that limited low-dose irradiation (LDI) using either γ-ray or x-ray exposure (0.4 Gy) significantly enhanced HR frequency, possibly through induction of DNA repair/recombination machinery including ataxia-telangiectasia mutated, histone H2A.X and RAD51 proteins. LDI could also increase HR efficiency by more than 30-fold when combined with the targeting tools zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats. Whole-exome sequencing confirmed that the LDI administered to hPSCs did not induce gross genomic alterations or affect cellular viability. Irradiated and targeted lines were karyotypically normal and made all differentiated lineages that continued to express green fluorescent protein targeted at the AAVS1 locus. This simple method allows higher throughput of new, targeted hPSC lines that are crucial to expand the use of disease modeling and to develop novel avenues of cell therapy. The simple and relevant technique described in this report uses a low level of radiation to increase desired gene modifications in human pluripotent stem cells by an order of magnitude. This higher efficiency permits greater throughput with reduced time and cost. The low level of radiation also greatly increased the recombination frequency when combined with developed engineered nucleases. Critically, the radiation did not lead to increases in DNA mutations or to reductions in overall cellular viability. This novel technique enables not only the rapid production of disease models using human stem cells but also the possibility of treating genetically based diseases by correcting patient-derived cells. ©AlphaMed Press.

Peri-operative management for excision of plexiform ...

African Journals Online (AJOL)

Case report: A 28-year-old female weighing 78 kilograms presented at the University of Port Harcourt Teaching Hospital (UPTH) with a huge left thigh mass, nodules and brownish skin patches (café-au-lait spots) all over her body. Plexiform neurofibromatosis was diagnosed. The mass was subsequently excised under ...

Base Sequence Context Effects on Nucleotide Excision Repair

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Yuqin Cai

2010-01-01

Full Text Available Nucleotide excision repair (NER plays a critical role in maintaining the integrity of the genome when damaged by bulky DNA lesions, since inefficient repair can cause mutations and human diseases notably cancer. The structural properties of DNA lesions that determine their relative susceptibilities to NER are therefore of great interest. As a model system, we have investigated the major mutagenic lesion derived from the environmental carcinogen benzo[a]pyrene (B[a]P, 10S (+-trans-anti-B[a]P-2-dG in six different sequence contexts that differ in how the lesion is positioned in relation to nearby guanine amino groups. We have obtained molecular structural data by NMR and MD simulations, bending properties from gel electrophoresis studies, and NER data obtained from human HeLa cell extracts for our six investigated sequence contexts. This model system suggests that disturbed Watson-Crick base pairing is a better recognition signal than a flexible bend, and that these can act in concert to provide an enhanced signal. Steric hinderance between the minor groove-aligned lesion and nearby guanine amino groups determines the exact nature of the disturbances. Both nearest neighbor and more distant neighbor sequence contexts have an impact. Regardless of the exact distortions, we hypothesize that they provide a local thermodynamic destabilization signal for repair.

Conservative treatment of rectal cancer with local excision and postoperative radiation therapy

International Nuclear Information System (INIS)

Minsky, B.D.

1995-01-01

The conventional surgical treatment for patients with potentially curable transmural and/or node positive rectal cancer is a low anterior resection or abdominoperineal resection. Recently, there has been increasing interest in the use of local excision and postoperative radiation therapy as primary therapy for selected rectal cancers. The limited data suggest that the approach of local excision and postoperative radiation therapy should be limited to patients with either T 1 tumours with adverse pathological factors or T 2 tumours. Transmural tumours, which have a 24% local failure rate, are treated more effectively with standard surgery and pre- or postoperative therapy. The results of local excision and postoperative radiation therapy are encouraging, but more experience is needed to determine if this approach ultimately has similar local control and survival rates as standard surgery. (author)

Identification and characterization of inhibitors of human apurinic/apyrimidinic endonuclease APE1.

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Anton Simeonov

2009-06-01

Full Text Available APE1 is the major nuclease for excising abasic (AP sites and particular 3'-obstructive termini from DNA, and is an integral participant in the base excision repair (BER pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC(1280, a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays -- a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen -- and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals.

Frontal recurrence of medulloblastoma five years after excision and craniospinal irradiation

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Roka Y

2009-07-01

Full Text Available Medulloblastomas were originally classified under gliomas of the cerebellum until Bailey and Cushing in 1925 named these tumors as medulloblastoma. At present these tumors are classified under primitive neuroectodermal tumor. Surgical excision followed by craniospinal irradiation is the treatment of choice. A 13-year-old-girl operated for posterior fossa medulloblastoma 5 years ago presented with history of headache and vomiting on and off for 4 days in late August 2008. The MRI showed left frontal tumor which on excision was reported as medulloblastoma. Even after optimal treatment reports of recurrence abound in literature. The most common location is in the posterior fossa, followed by spinal, supratentorial, and uncommonly, systemic metastases. We conclude that medulloblastomas are highly aggressive tumor with high local recurrences if the initial excision is incomplete and that recurrence in the supratentorial area although uncommon is still a possibility. This mandates regular follow up of these children till adulthood to catch early recurrences and metastatic disease.

Incomplete excision of non-melanoma skin cancer of the head and neck: can we predict failure?

DEFF Research Database (Denmark)

Kjerkegaard, Ulrik Knap; Stolle, Lars Bjørn

2014-01-01

included into the study. Patients were identified by the diagnostic (diagnosis-related group (DRG)) codes from DC44.0 to DC44.4. A total of 437 patients were treated for 516 skin lesions. Results Mean age was 71.4 years and the male–female ratio was 1.29. Incomplete tumor removal was found in 11 % of all...... for complete tumor excision is mandatory prior to reconstruction. Our findings showed that causes of incomplete excision could be identified. With this knowledge, we are able to optimize our quality of treatment, patient satisfaction, and finally, the cost/effectiveness of our department. Level of Evidence......Background Reported incomplete excision rates vary widely. This study described a single center's treatment of basal cell carcinoma (BCC) and squamous cell carcinomas (SCC) of the head and neck and investigated possible causes of incomplete excision. Methods All excised BCCs and SCCs in 2011 were...

Nucleotide excision repair II: From yeast to mammals

NARCIS (Netherlands)

J.H.J. Hoeijmakers (Jan)

1993-01-01

textabstractAn intricate network of repair systems safeguards the integrity of genetic material, by eliminating DNA lesions induced by numerous environmental and endogenous genotoxic agents. Nucleotide excision repair (NER) is one of the most versatile DNA repair systems. Deficiencies in this

Awake craniotomy for excision of arteriovenous malformations? A qualitative comparison study with stereotactic radiosurgery.

Science.gov (United States)

Chan, David Yuen Chung; Chan, Danny Tat Ming; Zhu, Cannon Xian Lun; Kan, Patricia Kwok Yee; Ng, Amelia Yikjin; Hsieh, Yi-Pin Sonia; Abrigo, Jill; Poon, Wai Sang; Wong, George Kwok Chu

2018-05-01

Treatment of arteriovenous malformations (AVM) located at the eloquent area has been a challenge. Awake brain mapping allows identification of a non-eloquent gyrus for intervention and can potentially facilitate resection with preservation of functions. An alternative treatment option is stereotactic radiosurgery (SRS). The objective of this study was to perform a qualitative comparison of the treatment outcome of awake AVM excision versus SRS. We conducted a 13-year retrospective review of AVM excision under awake craniotomy performed at Prince of Wales Hospital, Hong Kong, from 2003 to 2016. Patients' presentation, Spetzler-Martin (SM) grading, rate of obliteration and complication were reviewed and analyzed with the modified radiosurgery-based AVM score (RS score). Six patients had excision of AVM under awake mapping during this period of time. Two were SM Grade II and four were SM Grade III. Five located at the peri-rolandic region while one at the temporal language area. None had failed mapping. Five out of six achieved complete obliteration (83.3%). Qualitative comparative analysis had revealed better treatment outcome with awake AVM excision as compared to SRS with the obliteration rate of 100% versus 96% for RS score ≤1.00, 100% versus 78% for RS score 1.01-1.50, and 66% versus 50% for RS score >2.00 respectively. In conclusion, awake mapping and excision of AVMs at the eloquent area is feasible. Qualitative comparative analysis had revealed higher obliteration rate with awake AVM excision as compared to SRS. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

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Tatiana Flisikowska

Full Text Available 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.

Conserved XPB Core Structure and Motifs for DNA Unwinding:Implications for Pathway Selection of Transcription or ExcisionRepair

Energy Technology Data Exchange (ETDEWEB)

Fan, Li; Arval, Andrew S.; Cooper, Priscilla K.; Iwai, Shigenori; Hanaoka, Fumio; Tainer, John A.

2005-04-01

The human xeroderma pigmentosum group B (XPB) helicase is essential for transcription, nucleotide excision repair, and TFIIH functional assembly. Here, we determined crystal structures of an Archaeoglobus fulgidus XPB homolog (AfXPB) that characterize two RecA-like XPB helicase domains and discover a DNA damage recognition domain (DRD), a unique RED motif, a flexible thumb motif (ThM), and implied conformational changes within a conserved functional core. RED motif mutations dramatically reduce helicase activity, and the DRD and ThM, which flank the RED motif, appear structurally as well as functionally analogous to the MutS mismatch recognition and DNA polymerase thumb domains. Substrate specificity is altered by DNA damage, such that AfXPB unwinds dsDNA with 3' extensions, but not blunt-ended dsDNA, unless it contains a lesion, as shown for CPD or (6-4) photoproducts. Together, these results provide an unexpected mechanism of DNA unwinding with Implications for XPB damage verification in nucleotide excision repair.

Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining.

Science.gov (United States)

Moscariello, Mario; Wieloch, Radi; Kurosawa, Aya; Li, Fanghua; Adachi, Noritaka; Mladenov, Emil; Iliakis, George

2015-07-01

Exposure of cells to ionizing radiation or radiomimetic drugs generates DNA double-strand breaks that are processed either by homologous recombination repair (HRR), or by canonical, DNA-PKcs-dependent non-homologous end-joining (C-NHEJ). Chemical or genetic inactivation of factors involved in C-NHEJ or HRR, but also their local failure in repair proficient cells, promotes an alternative, error-prone end-joining pathway that serves as backup (A-EJ). There is evidence for the involvement of Artemis endonuclease, a protein deficient in a human radiosensitivity syndrome associated with severe immunodeficiency (RS-SCID), in the processing of subsets of DSBs by HRR or C-NHEJ. It is thought that within HRR or C-NHEJ Artemis processes DNA termini at complex DSBs. Whether Artemis has a role in A-EJ remains unknown. Here, we analyze using pulsed-field gel electrophoresis (PFGE) and specialized reporter assays, DSB repair in wild-type pre-B NALM-6 lymphocytes, as well as in their Artemis(-/-), DNA ligase 4(-/-) (LIG4(-/-)), and LIG4(-/-)/Artemis(-/-) double mutant counterparts, under conditions allowing evaluation of A-EJ. Our results substantiate the suggested roles of Artemis in C-NHEJ and HRR, but also demonstrate a role for the protein in A-EJ that is confirmed in Artemis deficient normal human fibroblasts. We conclude that Artemis is a nuclease participating in DSB repair by all major repair pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

Laparoscopic complete mesocolic excision via combined medial and cranial approaches for transverse colon cancer.

Science.gov (United States)

Mori, Shinichiro; Kita, Yoshiaki; Baba, Kenji; Yanagi, Masayuki; Tanabe, Kan; Uchikado, Yasuto; Kurahara, Hiroshi; Arigami, Takaaki; Uenosono, Yoshikazu; Mataki, Yuko; Okumura, Hiroshi; Nakajo, Akihiro; Maemura, Kosei; Natsugoe, Shoji

2017-05-01

To evaluate the safety and feasibility of laparoscopic complete mesocolic excision via combined medial and cranial approaches with three-dimensional visualization around the gastrocolic trunk and middle colic vessels for transverse colon cancer. We evaluated prospectively collected data of 30 consecutive patients who underwent laparoscopic complete mesocolic excision between January 2010 and December 2015, 6 of whom we excluded, leaving 24 for the analysis. We assessed the completeness of excision, operative data, pathological findings, length of large bowel resected, complications, length of hospital stay, and oncological outcomes. Complete mesocolic excision completeness was graded as the mesocolic and intramesocolic planes in 21 and 3 patients, respectively. Eleven, two, eight, and three patients had T1, T2, T3, and T4a tumors, respectively; none had lymph node metastases. A mean of 18.3 lymph nodes was retrieved, and a mean of 5.4 lymph nodes was retrieved around the origin of the MCV. The mean large bowel length was 21.9 cm, operative time 274 min, intraoperative blood loss 41 mL, and length of hospital stay 15 days. There were no intraoperative and two postoperative complications. Our procedure for laparoscopic complete mesocolic excision via combined medial and cranial approaches is safe and feasible for transverse colon cancer.

Important role of the nucleotide excision repair pathway in Mycobacterium smegmatis in conferring protection against commonly encountered DNA-damaging agents.

Science.gov (United States)

Kurthkoti, Krishna; Kumar, Pradeep; Jain, Ruchi; Varshney, Umesh

2008-09-01

Mycobacteria are an important group of human pathogens. Although the DNA repair mechanisms in mycobacteria are not well understood, these are vital for the pathogen's persistence in the host macrophages. In this study, we generated a null mutation in the uvrB gene of Mycobacterium smegmatis to allow us to compare the significance of the nucleotide excision repair (NER) pathway with two important base excision repair pathways, initiated by uracil DNA glycosylase (Ung) and formamidopyrimidine DNA glycosylase (Fpg or MutM), in an isogenic strain background. The strain deficient in NER was the most sensitive to commonly encountered DNA-damaging agents such as UV, low pH, reactive oxygen species, hypoxia, and was also sensitive to acidified nitrite. Taken together with previous observations on NER-deficient M. tuberculosis, these results suggest that NER is an important DNA repair pathway in mycobacteria.

Incidental intraductal papillomas (breast diagnosed on needle core biopsy do not need to be excised.

Science.gov (United States)

Jaffer, Shabnam; Bleiweiss, Ira J; Nagi, Chandandeep

2013-01-01

Most authors recommend excision of intraductal papillomas diagnosed on core needle biopsy. This leads to the question of whether or not excision is necessary for incidental intraductal papillomas on core needle biopsy as opposed to those corresponding to imaging findings. Using the pathology computerized data base we retrospectively identified 46 incidental intraductal papillomas diagnosed on core needle biopsy from 1/2000 to 12/2008. Clinical, radiologic, and pathologic information was gathered and correlated. All core needle biopsies were reviewed to confirm the diagnosis of incidental intraductal papillomas, and excision specimens reviewed when available. Of the 46 patients, follow-up information was available in only 38. The age of the patients ranged from 39 to 82 years (mean = 48 years). Most incidental intraductal papillomas were diagnosed by mammotome core needle biopsy (36 cases). A total of 33 cases were performed for calcifications with the following indications: clustered = 21, new = 4, pleomorphic = 3, increasing = 3, indeterminant = 2. The correlating diagnoses included the following: fibrocystic changes with calcium phosphate = 18 or calcium oxalate = 10, fibroadenoma with calcifications = 5. The three masses were: two cases of cystic papillary apocrine metaplasia (I Ultrasound and 1 MRI) and 1 fibroadenoma (Ultrasound). In all cases, the intraductal papillomas were ≤0.2 cm, were not associated with calcifications, and were incidental to them or the underlying mass. A total of 14 patients underwent excision, whereas the remaining 24 have remained radiologically stable for over 12 months. The excision specimen findings were: fibrocystic changes = 8 and intraductal papilloma = 6. With the exception of one case, all the intraductal papilloma remained incidental to imaging findings. In this solitary case, the calcifications were described as pleomorphic and corresponded to fibrocystic changes calcifications on core needle

Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification

KAUST Repository

Li, Lixin

2012-01-22

Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants. © 2012 Springer Science+Business Media B.V.

The influence of altered gravity on carbohydrate metabolism in excised wheat leaves

Science.gov (United States)

Obenland, D. M.; Brown, C. S.

1994-01-01

We developed a system to study the influence of altered gravity on carbohydrate metabolism in excised wheat leaves by means of clinorotation. The use of excised leaves in our clinostat studies offered a number of advantages over the use of whole plants, most important of which were minimization of exogenous mechanical stress and a greater amount of carbohydrate accumulation during the time of treatment. We found that horizontal clinorotation of excised wheat leaves resulted in significant reductions in the accumulation of fructose, sucrose, starch and fructan relative to control, vertically clinorotated leaves. Photosynthesis, dark respiration and the extractable activities of ADP glucose pyrophosphorylase (EC 2.7.7.27), sucrose phosphate synthase (EC 2.4.4.14), sucrose sucrose fructosyltransferase (EC 2.4.1.99), and fructan hydrolase (EC 3.2.1.80) were unchanged due to altered gravity treatment.

Syntheses of prodrug-type phosphotriester oligonucleotides responsive to intracellular reducing environment for improvement of cell membrane permeability and nuclease resistance.

Science.gov (United States)

Hayashi, Junsuke; Samezawa, Yusuke; Ochi, Yosuke; Wada, Shun-Ichi; Urata, Hidehito

2017-07-15

We synthesized prodrug-type phosphotriester (PTE) oligonucleotides containing the six-membered cyclic disulfide moiety by using phosphoramidite chemistry. Prodrug-type oligonucleotides named "Reducing-Environment-Dependent Uncatalyzed Chemical Transforming (REDUCT) PTE oligonucleotides" were converted into natural oligonucleotides under cytosol-mimetic reductive condition. Furthermore, the REDUCT PTE oligonucleotides were robust to nuclease digestion and exhibited good cell membrane permeability. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Transanal total mesorectal excision for rectal cancer - just a fashion trend?].

Science.gov (United States)

Kala, Z; Skrovina, M; Procházka, V; Grolich, T; Klos, K

2014-12-01

Transanal total mesorectal excision performed using equipment for transanal minimally invasive surgery is an innovative surgical technique introduced to facilitate this procedure and to reach better oncosurgical outcomes in patients with low rectal cancer. This article presents a brief summary of guidelines for treatment of patients with low rectal carcinoma. Up-to-date information about the principles of this new method, its modifications and contemporary indications is presented. Based on their own experience and literature resources, the authors inform about the advantages, limitations and unresolved issues of minimally invasive transanal mesorectal excision.

A child with xeroderma pigmentosum for excision of basal cell carcinoma

Directory of Open Access Journals (Sweden)

Sridevi M Mulimani

2013-01-01

Full Text Available Xeroderma pigmentosum (XP is characterized by hypersensitivity to sunlight, ocular involvement, and progressive neurological complications. These manifestations are due to a cellular hypersensitivity to ultraviolet radiation leading to a defect in repair of DNA by the process of nucleotide excision repair. Basal cell carcinoma which is rare in children can occur with XP. Though the XP induced changes are predominately dermatologic, pose several challenges in anaesthetic management. Hence, we are reporting a 9-year-old child with XP scheduled for excision of basal cell carcinoma under general anaesthesia.

The modification of siRNA with 3' cholesterol to increase nuclease protection and suppression of native mRNA by select siRNA polyplexes.

Science.gov (United States)

Ambardekar, Vishakha V; Han, Huai-Yun; Varney, Michelle L; Vinogradov, Serguei V; Singh, Rakesh K; Vetro, Joseph A

2011-02-01

Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3' cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC. Copyright © 2010 Elsevier Ltd. All rights reserved.

Endoscope-assisted approach to excision of branchial cleft cysts.

Science.gov (United States)

Teng, Stephanie E; Paul, Benjamin C; Brumm, John D; Fritz, Mark; Fang, Yixin; Myssiorek, David

2016-06-01

The purpose of this study is to describe an endoscope-assisted surgical technique for the excision of branchial cleft cysts and compare it to the standard approach. Retrospective case series review. Twenty-seven cases described as branchial cleft excisions performed by a single surgeon at one academic medical center were identified between 2007 and 2014. Twenty-five cases (8 endoscopic, 17 standard approach) were included in the study. Cases were excluded if final pathology was malignant. Patient charts were reviewed, and two techniques were compared through analysis of incision size, operative time, and surgical outcomes. This study showed that the length of incision required for the endoscopic approach (mean = 2.13 ± 0.23) was significantly less than that of the standard approach (mean = 4.10 ± 1.46, P = 0.008) despite the fact that there was no significant difference in cyst size between the two groups (P = 0.09). The other variables examined, including operative time and surgical outcomes, were not significantly different between the two groups. This transcervical endoscope-assisted approach to branchial cleft cyst excision is a viable option for uncomplicated cases. It provides better cosmetic results than the standard approach and does not negatively affect outcomes, increase operative time, or result in recurrence. 4. Laryngoscope, 126:1339-1342, 2016. © 2015 The American Laryngological, Rhinological and Otological Society, Inc.

Functional Interplay of the Mre11 Nuclease and Ku in the Response to Replication-Associated DNA Damage ▿

Science.gov (United States)

Foster, Steven S.; Balestrini, Alessia; Petrini, John H. J.

2011-01-01

The Mre11 complex is a central component of the DNA damage response, with roles in damage sensing, molecular bridging, and end resection. We have previously shown that in Saccharomyces cerevisiae, Ku70 (yKu70) deficiency reduces the ionizing radiation sensitivity of mre11Δ mutants. In this study, we show that yKu70 deficiency suppressed the camptothecin (CPT) and methyl methanesulfonate (MMS) sensitivity of nuclease-deficient mre11-3 and sae2Δ mutants in an Exo1-dependent manner. CPT-induced G2/M arrest, γ-H2AX persistence, and chromosome breaks were elevated in mre11-3 mutants. These outcomes were reduced by yKu70 deficiency. Given that the genotoxic effects of CPT are manifest during DNA replication, these data suggest that Ku limits Exo1-dependent double-strand break (DSB) resection during DNA replication, inhibiting the initial processing steps required for homology-directed repair. We propose that Mre11 nuclease- and Sae2-dependent DNA end processing, which initiates DSB resection prevents Ku from engaging DSBs, thus promoting Exo1-dependent resection. In agreement with this idea, we show that Ku affinity for binding to short single-stranded overhangs is much lower than for blunt DNA ends. Collectively, the data define a nonhomologous end joining (NHEJ)-independent, S-phase-specific function of the Ku heterodimer. PMID:21876003

Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2

Science.gov (United States)

Tsabar, Michael; Eapen, Vinay V.; Mason, Jennifer M.; Memisoglu, Gonen; Waterman, David P.; Long, Marcus J.; Bishop, Douglas K.; Haber, James E.

2015-01-01

In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5′ to 3′ end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5′ to 3′ resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells. PMID:26019182

Excise Taxes and the Price Elasticity of Demand.

Science.gov (United States)

Gamble, Ralph C., Jr.

1989-01-01

Points out that, although the analysis of the imposition of an excise tax is widely used in economics courses, the consequences of a change in the tax rate are different and ignored. This article presents an effective way to teach about such a change. (GG)

Abscisic Acid Accumulates at Positive Turgor Potential in Excised Soybean Seedling Growing Zones 1

Science.gov (United States)

Creelman, Robert A.; Mullet, John E.

1991-01-01

Abscisic acid (ABA) accumulated in soybean (Glycine max [L.] Merr. cv Williams) hypocotyl elongating regions when seedlings were transferred to low water potential vermiculite (Ψ = −0.3 megapascals) even though positive turgor is retained in this tissue. Accumulation of ABA in growing zones could occur from de novo biosynthesis within this tissue or transport from adjacent nongrowing zones. Both growing and nongrowing hypocotyl and root tissues accumulated significant levels of ABA when excised and dehydrated to reduce turgor. Surprisingly, excised growing zones (which experienced no water loss) also accumulated ABA when incubated in darkness for 4 hours at 100% relative humidity and 29°C. Induction of ABA accumulation in the excised elongating region of the hypocotyl was not caused by disruption of root pressure or wounding. While excision of hypocotyl elongating regions induced ABA accumulation, no change in either extensin or p33 mRNA levels was observed. Accumulation of extensin or p33 mRNA required more severe wounding. This suggests that ABA is not involved in the response of these genes in wounded tissue and that wound signals are not causing ABA accumulation in excised tissue. Accumulation of ABA in excised elongating regions was correlated with growth inhibition and a decline in turgor to the yield threshold (Ψ;p = 0.37 megapascals; R Matyssek, S Maruyama, JS Boyer [1988] Plant Physiol 86: 1163-1167). Inhibiting hypocotyl growth by transferring seedlings to lower temperatures or light did not cause ABA accumulation. We conclude that induction of ABA accumulation in growing zones is more sensitive to changes in turgor than the induction which occurs in mature tissues. Images Figure 2 PMID:16668113

Inhibition of excision repair of DNA in u.v.-irradiated Escherichia coli by phenethyl alcohol

International Nuclear Information System (INIS)

Tachibana, A.; Yonei, S.

1985-01-01

Membrane-specific drugs such as procaine and chlorpromazine have been shown to inhibit excision repair of DNA in u.v.-irradiated E. coli. One possible mechanism is that, if association of DNA with the cell membrane is essential for excision repair, this process may be susceptible to drugs affecting the structure of cell membranes. We examined the effect of phenethyl alcohol, which is a membrane-specific drug and known to dissociate the DNA-membrane complex, on excision repair of DNA in u.v.-irradiated E. coli cells. The cells were irradiated with u.v. light and then held at 30 0 C in buffer (liquid-holding) in the presence or absence of phenethyl alcohol. It was found that phenethyl alcohol inhibits the liquid-holding recovery in both wild-type and recA strains, corresponding to its dissociating action on the DNA-membrane complex. Thus, the association of DNA with cell membrane is an important factor for excision repair in E. coli. Procaine did not show the dissociating effect, suggesting that at least two different mechanisms are responsible for the involvement of cell membrane in excision repair of DNA in E. coli. (author)

Increased cell division but not thymic dysfunction rapidly affects the T-cell receptor excision circle content of the naive T cell population in HIV-1 infection

NARCIS (Netherlands)

Hazenberg, Mette D.; Borleffs, J.C.C.; Otto, S.A.; Cohen Stuart, J.W.T. (James Willem Theodoor); Verschuren, M.C.M. (Martie); Boucher, C.A.B.; Coutinho, R.A.; Lange, Joep M.A.; Rinke de Wit, T.F. (Tobias); Tsegaye, A. (Aster); Dongen, J.J.M. (Jaques) van; Hamann, D. (Dörte); Boer, R.J. de; Miedema, F.

2000-01-01

Recent thymic emigrants can be identified by T cell receptor excision circles (TRECs) formed during T-cell receptor rearrangement. Decreasing numbers of TRECs have been observed with aging and in human immunodeficiency virus (HIV)-1 infected individuals, suggesting for thymic impairment. Here,

Characterization of the residual structure in the unfolded state of the Delta 131 Delta fragment of staphylococcal nuclease

DEFF Research Database (Denmark)

Francis, C. J.; Lindorff-Larsen, Kresten; Best, R. B.

2006-01-01

dynamics simulations to characterise the residual structure of the 131 fragment of staphylococcal nuclease under physiological conditions. Our findings indicate that 131 under these conditions shows a tendency to form transiently hydrophobic clusters similar to those present in the native state of wild......The determination of the conformational preferences in unfolded states of proteins constitutes an important challenge in structural biology. We use inter-residue distances estimated from site-directed spin-labeling NMR experimental measurements as ensemble-averaged restraints in all-atom molecular...

Transanal vs laparoscopic total mesorectal excision for rectal cancer

DEFF Research Database (Denmark)

Perdawood, Sharaf; Al Khefagie, Ghalib Ali Abod

2016-01-01

BACKGROUND: Laparoscopic total mesorectal excision (LaTME) has improved short-term outcomes of rectal cancer surgery with comparable oncological results to open approach. LaTME can be difficult in the lower most part of the rectum, leading potentially to higher rates of complications, conversion...... to open surgery and probably suboptimal oncological quality. Transanal TME (TaTME) can potentially solve these problems. The aim of this study was to compare the short-term results after TaTME with those after LaTME. METHODS: A prospectively collected database of consecutive patients who underwent Ta......TME was maintained. Results were compared with those underwent LaTME in the preceding period. Patients who underwent low anterior resection or intersphincteric abdominoperineal excision (APE) were included. Primary end-points were radical resection and specimen quality. Secondary end-points were complications, rates...

Functionalized bioengineered spider silk spheres improve nuclease resistance and activity of oligonucleotide therapeutics providing a strategy for cancer treatment.

Science.gov (United States)

Kozlowska, Anna Karolina; Florczak, Anna; Smialek, Maciej; Dondajewska, Ewelina; Mackiewicz, Andrzej; Kortylewski, Marcin; Dams-Kozlowska, Hanna

2017-09-01

Cell-selective delivery and sensitivity to serum nucleases remain major hurdles to the clinical application of RNA-based oligonucleotide therapeutics, such as siRNA. Spider silk shows great potential as a biomaterial due to its biocompatibility and biodegradability. Self-assembling properties of silk proteins allow for processing into several different morphologies such as fibers, scaffolds, films, hydrogels, capsules and spheres. Moreover, bioengineering of spider silk protein sequences can functionalize silk by adding peptide moieties with specific features including binding or cell recognition domains. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics, such as CpG-siRNA. The MS2 bioengineered silk was functionalized with poly-lysine domain (KN) to generate hybrid silk MS2KN. CpG-siRNA efficiently bound to MS2KN in contrary to control MS2. Both MS2KN complexes and spheres protected CpG-siRNA from degradation by serum nucleases. CpG-siRNA molecules encapsulated into MS2KN spheres were efficiently internalized and processed by TLR9-positive macrophages. Importantly, CpG-STAT3siRNA loaded in silk spheres showed delayed and extended target gene silencing compared to naked oligonucleotides. The prolonged Stat3 silencing resulted in the more pronounced downregulation of interleukin 6 (IL-6), a proinflammatory cytokine and upstream activator of STAT3, which limits the efficacy of TLR9 immunostimulation. Our results demonstrate the feasibility of using spider silk spheres as a carrier of therapeutic nucleic acids. Moreover, the modified kinetic and activity of the CpG-STAT3siRNA embedded into silk spheres is likely to improve immunotherapeutic effects in vivo. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel

Coronary artery bypass grafting and concomitant excision of chest wall chondrosarcoma

Directory of Open Access Journals (Sweden)

Ganti Somsekhar

2009-02-01

Full Text Available Abstract Coexistence of coronary artery disease and cancer with both requiring surgical treatment at the same time is rare. A 52 year male undergoing elective coronary artery bypass grafting was incidentally discovered to have a large soft tissue mass of variable consistency with cartilaginous elements arising from the right costal margin and adjoining ribs by a broad attachment and protruding into right pleural cavity. Frozen section suggested it to be either a chondrosarcoma or a teratoma. A wide excision of the mass with the adjoining muscle and periosteum along with quadruple coronary artery bypass grafting was done. This report is unusual on account of a being the first reported case in world literature of concomitant excision of chondrosarcoma and coronary artery bypass grafting and b the conservative management of the incidentally discovered chondrosarcoma by wide excision rather than chest wall resection with no local recurrence to date. Pathology of chondrosarcoma, in particular, and various management strategies when coronary artery disease and cancer coexist, in general, is discussed.

Nail-fold excision for the treatment of ingrown toenail in children.

Science.gov (United States)

Haricharan, Ramanath N; Masquijo, Javier; Bettolli, Marcos

2013-02-01

To evaluate the effectiveness of the nail-fold excision procedure in children. Prospectively collected data on patients less than 18 years of age who underwent a nail-fold excision for symptomatic ingrown toenail were analyzed. Patients were seen in 2 centers and data collected included demographics, site of ingrown toenail, complications (including recurrence), patient satisfaction, and duration of follow-up. Overall, 67 procedures were performed on 50 patients between June 2009 and July 2011 at the 2 institutions. The mean age was 14 years (range, 9-18 years) and 30 were male patients. No recurrences were seen after a follow-up for a median of 14 months (range 6-28 months). Patients were very satisfied with the cosmetic outcomes. Six minor complications occurred, including 3 patients with bleeding requiring dressing change, 2 with excessive granulation tissue, and 1 with nail growth abnormality. The nail-fold excision technique is highly effective in the pediatric population, with no recurrence, excellent cosmesis, and very high patient satisfaction. Copyright © 2013 Mosby, Inc. All rights reserved.

Autobiographical memory of the recent past following frontal cortex or temporal lobe excisions.

Science.gov (United States)

Thaiss, Laila; Petrides, Michael

2008-08-01

Previous research has raised questions regarding the necessity of the frontal cortex in autobiographical memory and the role that it plays in actively retrieving contextual information associated with personally relevant events. Autobiographical memory was studied in patients with unilateral excisions restricted to the frontal cortex or temporal lobe involving the amygdalo-hippocampal region and in normal controls using an event-sampling method. We examined accuracy of free recall, use of strategies during retrieval and memory for specific aspects of the autobiographical events, including temporal order. Patients with temporal lobe excisions were impaired in autobiographical recall. By contrast, patients with frontal cortical excisions exhibited normal autobiographical recall but were less likely to use temporal order spontaneously to organize event retrieval. Instruction to organize retrieval by temporal order failed to improve recall in temporal lobe patients and increased the incidence of plausible intrusion errors in left temporal patients. In contrast, patients with frontal cortical excisions now surpassed control subjects in recall of autobiographical events. Furthermore, the retrieval accuracy for the temporal order of diary events was not impaired in these patients. In a subsequent cued recall test, temporal lobe patients were impaired in their memory for the details of the diary events and their context. In conclusion, a basic impairment in autobiographical memory (including memory for temporal context) results from damage to the temporal lobe and not the frontal cortex. Patients with frontal excisions fail to use organizational strategies spontaneously to aid retrieval but can use these effectively if instructed to do so.

Functional identification of the non-specific nuclease from white spot syndrome virus

International Nuclear Information System (INIS)

Li Li; Lin Shumei; Yanga Feng

2005-01-01

The product encoded by the wsv191 gene from shrimp white spot syndrome virus (WSSV) is homologous with non-specific nucleases (NSN) of other organisms. To functionally identify the protein, the wsv191 gene was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein with 6His-tag at C-terminal. The fusion protein (termed as rWSSV-NSN) was purified using Ni-NTA affinity chromatography under denatured conditions, renatured and characterized by three methods. The results showed that rWSSV-NSN could hydrolyze both DNA and RNA. 5'-RACE result revealed that the transcription initiation site of the wsv191 gene was located at nucleotide residue G of the predicted ATG triplet. Therefore, we concluded that the next ATG should be the genuine translation initiation codon of the wsv191 gene. Western blot analysis revealed that the molecular mass of natural WSSV-NSN was 37 kDa

Excision repair in ataxia telangiectasia, Fanconi's anemia, Cockayne syndrome, and Bloom's syndrome after treatment with ultraviolet radiation and N-acetoxy-2-acetylaminofluorene

International Nuclear Information System (INIS)

Ahmed, F.E.; Setlow, R.B.

1978-01-01

Excision repair of damage due to ultraviolet radiation, N-acetoxy-2-acetylaminofluorene and a combination of both agents was studied in normal human fibroblasts and various cells from cancer prone patients (ataxia telangiectasia, Fanconi's anemia, Cockayne syndrome and Bloom's syndrome). Three methods giving similar results were used: unscheduled DNA synthesis by radioautography, photolysis of bromodeoxyuridine incorporated into parental DNA during repair, and loss of sites sensitive to an ultraviolet endonuclease. All cell lines were proficient in repair of ultraviolet and acetoxy acetylaminofluorene damage and at saturation doses of both agents repair was additive. We interpret these data as indicating that the rate limiting step in excision repair of ultraviolet and acetoxy acetylaminofluorene is different and that there are different enzyme(s) working on incision of both types of damages. (Auth.)

Increased Ac excision (iae): Arabidopsis thaliana mutations affecting Ac transposition

International Nuclear Information System (INIS)

Jarvis, P.; Belzile, F.; Page, T.; Dean, C.

1997-01-01

The maize transposable element Ac is highly active in the heterologous hosts tobacco and tomato, but shows very much reduced levels of activity in Arabidopsis. A mutagenesis experiment was undertaken with the aim of identifying Arabidopsis host factors responsible for the observed low levels of Ac activity. Seed from a line carrying a single copy of the Ac element inserted into the streptomycin phosphotransferase (SPT) reporter fusion, and which displayed typically low levels of Ac activity, were mutagenized using gamma rays. Nineteen mutants displaying high levels of somatic Ac activity, as judged by their highly variegated phenotypes, were isolated after screening the M2 generation on streptomycin-containing medium. The mutations fall into two complementation groups, iae1 and iae2, are unlinked to the SPT::Ac locus and segregate in a Mendelian fashion. The iae1 mutation is recessive and the iae2 mutation is semi-dominant. The iae1 and iae2 mutants show 550- and 70-fold increases, respectively, in the average number of Ac excision sectors per cotyledon. The IAE1 locus maps to chromosome 2, whereas the SPT::Ac reporter maps to chromosome 3. A molecular study of Ac activity in the iae1 mutant confirmed the very high levels of Ac excision predicted using the phenotypic assay, but revealed only low levels of Ac re-insertion. Analyses of germinal transposition in the iae1 mutant demonstrated an average germinal excision frequency of 3% and a frequency of independent Ac re-insertions following germinal excision of 22%. The iae mutants represents a possible means of improving the efficiency of Ac/Ds transposon tagging systems in Arabidopsis, and will enable the dissection of host involvement in Ac transposition and the mechanisms employed for controlling transposable element activity

Symptomatic pancreatic heterotopia treated by local excision.

Science.gov (United States)

De Friend, D J; Saa-Gandi, F W; Humphrey, C S; Foster, D N

1991-01-01

Non-ulcer dyspepsia is a continuing problem and in many cases a precise cause is never identified. We present five patients with an allegedly uncommon condition--pancreatic heterotopia. They were managed by local excision of the tumour and after a mean (range) follow up of 42 (9-80) months all remain free of the original symptoms. Images Figure 1 Figure 2 PMID:2013433

May I Cut in? Gene Editing Approaches in Human Induced Pluripotent Stem Cells.

Science.gov (United States)

Brookhouser, Nicholas; Raman, Sreedevi; Potts, Christopher; Brafman, David A

2017-02-06

In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies have further accelerated the potential of hiPSCs. In this review, we discuss the various gene editing technologies that are being implemented with hiPSCs. Specifically, we describe the emergence of technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 that can be used to edit the genome at precise locations, and discuss the strengths and weaknesses of each of these technologies. In addition, we present the current applications of these technologies in elucidating the mechanisms of human development and disease, developing novel and effective therapeutic molecules, and engineering cell-based therapies. Finally, we discuss the emerging technological advances in targeted gene editing methods.

Histone displacement during nucleotide excision repair

DEFF Research Database (Denmark)

Dinant, C.; Bartek, J.; Bekker-Jensen, S.

2012-01-01

Nucleotide excision repair (NER) is an important DNA repair mechanism required for cellular resistance against UV light and toxic chemicals such as those found in tobacco smoke. In living cells, NER efficiently detects and removes DNA lesions within the large nuclear macromolecular complex called...... of histone variants and histone displacement (including nucleosome sliding). Here we review current knowledge, and speculate about current unknowns, regarding those chromatin remodeling activities that physically displace histones before, during and after NER....

Impacts of restructuring tabacco excise tax in the Philippines

International Development Research Centre (IDRC) Digital Library (Canada)

A recently approved law restructuring tobacco taxes in the. Philippines includes a steep increase in the excise tax (the tax paid by producers which ... part of Canada's International Development Research Centre. (IDRC), a Canadian Crown ...

Design, construction, and analysis of specific zinc finger nucleases for microphthalmia - associate transcription factor

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Wenwen Wang

2012-08-01

Full Text Available This work studied the design, construction, and cleavage analysis of zinc finger nucleases (ZFNs that could cut the specific sequences within microphthalmia - associate transcription factor (mitfa of zebra fish. The target site and ZFPs were selected and designed with zinc finger tools, while the ZFPs were synthesized using DNAWorks and two-step PCR. The ZFNs were constructed, expressed, purified, and analyzed in vitro. As expected, the designed ZFNs could create a double-stand break (DSB at the target site in vitro. The DNAWorks, two-step PCR, and an optimized process of protein expression were firstly induced in the construction of ZFNs successfully, which was an effective and simplified protocol. These results could be useful for further application of ZFNs - mediated gene targeting.

A human repair gene ERCC5 is involved in group G xeroderma pigmentosum

International Nuclear Information System (INIS)

Shiomi, Tadahiro

1994-01-01

In E. coli, ultraviolet-induced DNA damage is removed by the coordinated action of UVR A, B, C, and D proteins (1). In Saccharomyces cerevisiae, more than ten genes have been reported to be involved in excision repair (2). The nucleotide excision repair pathway has been extensively studied in these organisms. To facilitate studying nucleotide excision repair in mammalian cells. Ultraviolet-sensitive rodent cell mutants have been isolated and classified into 11 complementation groups (9,10). The human nucleotide excision repair genes which complement the defects of the mutants have been designated as the ERCC (excision repair cross-complementing) genes; a number is added to refer to the particular rodent complementation group that is corrected by the gene. Recently, several human DNA repair genes have been cloned using rodent cell lines sensitive to ultraviolet. These include ERCC2 (3), ERCC3 (4), and ERCC6 (5), which correspond to the defective genes in the ultraviolet-sensitive human disorders xeroderma pigmentosum (XP) group D (6) and group B (4), and Cockayne's syndrome (CS) group B (7), respectively. The human excision repair gene ERCC5 was cloned after DNA-mediated gene transfer of human HeLa cell genomic DNA into the ultraviolet-sensitive mouse mutant XL216, a member of rodent complementation group 5 (11,12) and the gene was mapped on human chromosome 13q32.3-q33.1 by the replication R-banding fluorescence in situ hybridization method (13). The ERCC5 cDNA encodes a predicted 133 kDa nuclear protein that shares some homology with product of the yeast DNA repair gene RAD 2. Transfection with mouse ERCC5 cDNA restored normal levels of ultraviolet-resistance to XL216 cells. Microinjection of ERCC5 cDNA specifically restored the defect of XP group G cells (XP-G) as measured by unscheduled DNA synthesis (UDS), and XP-G cells stably transformed with ERCC5 cDNA showed nearly normal ultraviolet resistance. (J.P.N.)

Correlation of open cell-attached and excised patch clamp techniques.

Science.gov (United States)

Filipovic, D; Hayslett, J P

1995-11-01

The excised patch clamp configuration provides a unique technique for some types of single channel analyses, but maintenance of stable, long-lasting preparations may be confounded by rundown and/or rapid loss of seal. Studies were performed on the amiloride-sensitive Na+ channel, located on the apical surface of A6 cells, to determine whether the nystatin-induced open cell-attached patch could serve as an alternative configuration. Compared to excised inside-out patches, stable preparations were achieved more readily with the open cell-attached patch (9% vs. 56% of attempts). In both preparations, the current voltage (I-V) relation was linear, current amplitudes were equal at opposite equivalent clamped voltages, and Erev was zero in symmetrical Na+ solutions, indicating similar Na+ activities on the cytosolic and external surfaces of the patch. Moreover, there was no evidence that nystatin altered channel activity in the patch because slope conductance (3-4pS) and Erev (75 mV), when the bath was perfused with a high K:low Na solution (ENa = 80 mV), were nearly equal in both patch configurations. Our results therefore indicate that the nystatin-induced open cell-attached patch can serve as an alternative approach to the excised inside-out patch when experiments require modulation of univalent ions in the cytosol.

A retrospective study comparing the accuracy of prehistology diagnosis and surgical excision of malignant melanomas by general practitioners and hospital specialists.

Science.gov (United States)

Bakhai, M; Hopster, D; Wakeel, R

2010-01-01

A retrospective study was carried out to compare the overall standard of surgical excision of malignant melanomas (MMs) between general practitioners (GPs) and hospital specialists before and after the introduction of the UK melanoma guidelines between 1989 and 2006. In total, 213 melanoma excision reports were examined and surgical excision margins recorded. The results showed a significant difference in the rate of adequate surgical excision margins (at all levels of Breslow thickness) between GPs and hospital specialists, with hospital specialists excising melanomas with safe surgical excision margins at a significantly higher rate compared with GPs. Since the introduction of the guidelines in 2002, GPs showed a significant improvement in the completeness of melanoma excision but remained poor at prehistology diagnosis and in particular at taking adequate excision margins. Implementation of the guidelines has not produced significant improvements in adequacy of excision margins in both primary and secondary care. The results show that hospital specialists maintained a high standard of prehistological diagnosis and completeness of excision throughout the time of the study, performing at a significantly higher standard compared with GPs. Our conclusions concur with the UK melanoma guidelines and the National Institute for Health and Clinical Excellence guidelines, which suggest that lesions suspicious for melanoma should be urgently referred to a dermatologist or plastic surgeon for surgical excision and should not be surgically excised in primary care, particularly if lesions have a Breslow thickness > 2 mm. We suggest that the new guidelines need to be more aggressively implemented in primary care and guidance introduced to improve the accuracy of diagnosis, with better training provided for GPs.

Incidence of Incomplete Excision in Surgically Treated Cutaneous Squamous Cell Carcinoma and Identification of the Related Risk Factors

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Sara Sabouri Rad

2011-12-01

Full Text Available Squamous cell carcinoma (SCC is the second most common type of skin cancer with potential risks for metastasis and recurrence if left untreated or incompletely excised. This case series study was designed to determine the frequency of incompletely excised SCCs and the related risk factors. A total of 273 SCCs (253 patients excised in Razi dermatology hospital of Tehran from 2006-2008, were evaluated and were analyzed by Chi-square or t-test. The incidence of incomplete excision was 17.58 % and deep margin involvement was observed in 73% of lesions. Risk factors associated with incomplete excision of SCCs were being female, location of the tumors (in particular the lesions on lateral canthus, upper lip, foot, forehead, cheek, neck, nose and ear, large lesions and grafting method of repair. There was no statistically significant difference for the age, degree of histological differentiation, childhood history of radiotherapy for tinea capitis and the type of anesthesia. More care should be taken for high risk SCCs as complete excision avoids potential risk of recurrence and metastasis.

Periprosthetic soft tissue recurrence of chondroblastoma after attempted en bloc excision from the proximal humerus

Energy Technology Data Exchange (ETDEWEB)

Hammond, G.W.; Tehranzadeh, J.; Hoang, B.H. [University of California, Irvine, Department of Orthopaedic Surgery, Orange (United States); Gu, M. [University of California, Irvine, Department of Pathology, Orange (United States)

2006-01-01

A case of soft tissue recurrence of chondroblastoma after attempted en bloc excision and endoprosthetic replacement is described. This tumor in the proximal humerus recurred after initial curettage and was subsequently treated by attempted en bloc excision with positive microscopic margins. The patient then presented with a large soft tissue recurrence surrounding the endoprosthesis. This periprosthetic recurrence necessitated re-excision and revision of the endoprosthesis. Recurrence is not uncommon following curettage of chondroblastoma. However, less is known about soft tissue recurrence after en bloc resection of this tumor with positive margins. A subset of chondroblastoma may exist with more locally aggressive behavior. (orig.)

Excision repair of 5,6-dihydroxydihydrothymine from the DNA of Micrococcus radiodurans

International Nuclear Information System (INIS)

Targovnik, H.S.; Hariharan, P.V.

1980-01-01

One of the major ionizing radiation products, 5,6-dihydroxydihydrothymine (thymine glycol), was measured in the DNA of Micrococcus radiodurans following exposure of cells to 6.8-MeV electrons or 254-nm ultraviolet light. Removal of 5,6-dihydroxydihydrothymine was measured in both an ionizing radiation-sensitive strain (262) and a highly radioresistant strain (the wild type W + ) of Micrococcus radiodurans. Within 30 min of incubation (33 0 C) following exposure to ultraviolet light (2400 J/m 2 ) approximately 60% of the thymine glycols were excised, whereas in the case of ionizing radiation (250 krad) only 35% were removed from the cellular DNA of the wild-type strain. In contrast less than 50% of the thymine glycols were excised from the sensitive strain. The amount of DNA degradation induced by radiation was less than 10% in both strains. The results suggest a possible correlation between reduced excision repair of base damage and increased radiation sensitivity

Surgical pathology of excised heart valves in a referral hospital in iran

International Nuclear Information System (INIS)

Yaghoubi, A.R.; Raeesi, K.

2007-01-01

Assessment of surgical pathology of excised heart valves in a referral hospital in Iran in a five years period. This retrospective descriptive study was done from 2002 to 2005 in Rajaie heart center in Tehran, Iran. Surgery and pathology records of patients who underwent valve replacement or repair surgery were reviewed. Of 1563 patients 738 (47.2%) underwent mitral, 565 (36.1%) aortic, and 215 (14%) multivalve operation. Most common pathology of mitral valve was rheumatic (68%), while degenerative calcific pathology was dominant in aortic valve (52%). Rheumatic involvement was 46%, and degenerative pathology was common in tricuspid and pulmonary valves (50% and 67%, respectively). Time trend analysis shows no significant variation in excised valves pathology or pattern from 2002 to 2005 (p=0.112). Rheumatic pathology in excised heart valves is still common in this referral heart center in Iran, and no obvious change in this pattern was found during a 5 years period. (author)

Efficient methods for targeted mutagenesis in zebrafish using zinc-finger nucleases: data from targeting of nine genes using CompoZr or CoDA ZFNs.

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Raman Sood

Full Text Available Recently, it has been shown that targeted mutagenesis using zinc-finger nucleases (ZFNs and transcription activator-like effector nucleases (TALENs can be used to generate knockout zebrafish lines for analysis of their function and/or developing disease models. A number of different methods have been developed for the design and assembly of gene-specific ZFNs and TALENs, making them easily available to most zebrafish researchers. Regardless of the choice of targeting nuclease, the process of generating mutant fish is similar. It is a time-consuming and multi-step process that can benefit significantly from development of efficient high throughput methods. In this study, we used ZFNs assembled through either the CompoZr (Sigma-Aldrich or the CoDA (context-dependent assembly platforms to generate mutant zebrafish for nine genes. We report our improved high throughput methods for 1 evaluation of ZFNs activity by somatic lesion analysis using colony PCR, eliminating the need for plasmid DNA extractions from a large number of clones, and 2 a sensitive founder screening strategy using fluorescent PCR with PIG-tailed primers that eliminates the stutter bands and accurately identifies even single nucleotide insertions and deletions. Using these protocols, we have generated multiple mutant alleles for seven genes, five of which were targeted with CompoZr ZFNs and two with CoDA ZFNs. Our data also revealed that at least five-fold higher mRNA dose was required to achieve mutagenesis with CoDA ZFNs than with CompoZr ZFNs, and their somatic lesion frequency was lower (<5% when compared to CopmoZr ZFNs (9-98%. This work provides high throughput protocols for efficient generation of zebrafish mutants using ZFNs and TALENs.

The Modification of siRNA with 3′ Cholesterol to Increase Nuclease Protection and Suppression of Native mRNA by Select siRNA Polyplexes

Science.gov (United States)

Ambardekar, Vishakha V.; Han, Huai-Yun; Varney, Michelle L.; Vinogradov, Serguei V.; Singh, Rakesh K.; Vetro, Joseph A.

2010-01-01

Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3′ cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC. PMID:21047680

Physico-chemical and biological study of excision-repair of UV-irradiated PHIX 174 RF DNA in vitro

International Nuclear Information System (INIS)

Heijneker, H.L.

1975-01-01

A study is presented on the excision repair of ultraviolet-irradiated PHIX 174 RFI DNA in vitro with UV-specific endonuclease from micrococcus luteus, DNA polymerase I from E. coli and DNA ligase from phage T 4 infected E. coli. Excision repair was measured by physico-chemical and by biological methods. It is shown that more than 90% of the pyrimidine dimers can be repaired in vitro and that the repaired molecules have regained full biological activity. Endonuclease III was not essential for excision repair in vitro and did not stimulate repair; from this it was concluded that UV-endo generates 3' OH endgroups. The usefulness of the methods with regard to the study of excision repair is discussed

Sentinel node biopsy and concomitant probe-guided tumor excision of nonpalpable breast cancer.

Science.gov (United States)

van Rijk, Maartje C; Tanis, Pieter J; Nieweg, Omgo E; Loo, Claudette E; Olmos, Renato A Valdés; Oldenburg, Hester S A; Rutgers, Emiel J Th; Hoefnagel, Cornelis A; Kroon, Bin B R

2007-02-01

Preliminary data have shown encouraging results of a single intratumoral radiopharmaceutical injection that enables both sentinel node biopsy and probe-guided excision of the primary tumor in patients with nonpalpable breast cancer. The aim of the study was to evaluate this approach in a large group of patients. Lymphoscintigraphy was performed in 368 patients with nonpalpable breast cancer after intratumoral injection of (99m)Tc-nanocolloid (.2 mL, 123 MBq, 3.3 mCi) guided by ultrasound or stereotaxis. The sentinel node was pursued with the aid of vital blue dye (1.0 mL, intratumoral) and a gamma ray detection probe. In case of breast-conserving surgery, the probe was used to guide the excision. At least one sentinel node could be identified intraoperatively in 357 patients (97%), of whom 69 had involved nodes (19%). Age over 60 years was associated with less frequent nonaxillary lymphatic drainage and absence of internal mammary chain dissemination. Tumor-free margins were obtained in 262 (89%) of the 293 patients who underwent segmental excision. Re-excision of the primary tumor bed was performed in six patients (2%). During a median follow-up of 22 months, one breast recurrence and one axillary recurrence were observed. Lymphatic mapping and probe-guided tumor excision of nonpalpable breast cancer by intralesional administration of a single dose of (99m)Tc-nanocolloid and blue dye resulted in 97% identification of the sentinel node and in tumor-free margins in 89% of the patients who underwent breast-conserving surgery. Longer follow-up is needed to substantiate the accuracy and safety of this technique.

Isolating DNA from sexual assault cases: a comparison of standard methods with a nuclease-based approach

Science.gov (United States)

2012-01-01

Background Profiling sperm DNA present on vaginal swabs taken from rape victims often contributes to identifying and incarcerating rapists. Large amounts of the victim’s epithelial cells contaminate the sperm present on swabs, however, and complicate this process. The standard method for obtaining relatively pure sperm DNA from a vaginal swab is to digest the epithelial cells with Proteinase K in order to solubilize the victim’s DNA, and to then physically separate the soluble DNA from the intact sperm by pelleting the sperm, removing the victim’s fraction, and repeatedly washing the sperm pellet. An alternative approach that does not require washing steps is to digest with Proteinase K, pellet the sperm, remove the victim’s fraction, and then digest the residual victim’s DNA with a nuclease. Methods The nuclease approach has been commercialized in a product, the Erase Sperm Isolation Kit (PTC Labs, Columbia, MO, USA), and five crime laboratories have tested it on semen-spiked female buccal swabs in a direct comparison with their standard methods. Comparisons have also been performed on timed post-coital vaginal swabs and evidence collected from sexual assault cases. Results For the semen-spiked buccal swabs, Erase outperformed the standard methods in all five laboratories and in most cases was able to provide a clean male profile from buccal swabs spiked with only 1,500 sperm. The vaginal swabs taken after consensual sex and the evidence collected from rape victims showed a similar pattern of Erase providing superior profiles. Conclusions In all samples tested, STR profiles of the male DNA fractions obtained with Erase were as good as or better than those obtained using the standard methods. PMID:23211019

A tailored biocatalyst achieved by the rational anchoring of imidazole groups on a natural polymer: furnishing a potential artificial nuclease by sustainable materials engineering.

Science.gov (United States)

Ferreira, José G L; Grein-Iankovski, Aline; Oliveira, Marco A S; Simas-Tosin, Fernanda F; Riegel-Vidotti, Izabel C; Orth, Elisa S

2015-04-11

Foreseeing the development of artificial enzymes by sustainable materials engineering, we rationally anchored reactive imidazole groups on gum arabic, a natural biocompatible polymer. The tailored biocatalyst GAIMZ demonstrated catalytic activity (>10(5)-fold) in dephosphorylation reactions with recyclable features and was effective in cleaving plasmid DNA, comprising a potential artificial nuclease.

Risk and outcome analysis of 1832 consecutively excised basal cell carcinomas in a tertiary referral plastic surgery unit.

LENUS (Irish Health Repository)

Malik, Vinod

2012-02-01

BACKGROUND: Basal cell carcinomas are the most prevalent of all skin cancers worldwide and form the majority of the surgical workload for most modern cutaneous malignancy centres. Primary surgical removal of basal cell carcinomas remains the gold standard of treatment but, despite almost two centuries of surgical experience, rates of incomplete surgical excision of up to 50% are still reported. The aim of this study was to assess, quantify and perform comparative analysis of the outcomes and predictive factors of consecutive primarily-excised basal cell carcinomas in a tertiary centre over a six-year period. METHODS: Retrospective audit was conducted on all patients who underwent surgical excision of basal cell carcinomas from January 2000 to December 2005. Assessment parameters included patient biographics, tumour management differences and detailed histopathological analysis of tumour margins and subtypes. RESULTS: One thousand eight hundred and thirty two basal cell carcinomas were excised from 1329 patients over the designated time period. Two hundred and fifty one (14%) lesions were incompletely excised with 135 (7.4%) involving the peripheral margin only, 48 (2.6%) the deep margin only and 41 (2.2%) involving both. Nasal location was the most common predictor of incomplete excision. CONCLUSIONS: Overall basal cell carcinomas excision rates compared favourably with international reported standards but attention to a variety of surgical and histological risk factors may improve this further.

Interference Excision in Spread Spectrum Communications Using Adaptive Positive Time-Frequency Analysis

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Krishnan Sridhar

2007-01-01

Full Text Available This paper introduces a novel algorithm to excise single and multicomponent chirp-like interferences in direct sequence spread spectrum (DSSS communications. The excision algorithm consists of two stages: adaptive signal decomposition stage and directional element detection stage based on the Hough-Radon transform (HRT. Initially, the received spread spectrum signal is decomposed into its time-frequency (TF functions using an adaptive signal decomposition algorithm, and the resulting TF functions are mapped onto the TF plane. We then use a line detection algorithm based on the HRT that operates on the image of the TF plane and detects energy varying directional elements that satisfy a parametric constraint. Interference is modeled by reconstructing the corresponding TF functions detected by the HRT, and subtracted from the received signal. The proposed technique has two main advantages: (i it localizes the interferences on the TF plane with no cross-terms, thus facilitating simple filtering techniques based on thresholding of the TF functions, and is an efficient way to excise the interference; (ii it can be used for the detection of any directional interferences that can be parameterized. Simulation results with synthetic models have shown successful performance with linear and quadratic chirp interferences for single and multicomponent interference cases. The proposed method excises the interference even under very low SNR conditions of  dB, and the technique could be easily extended to any interferences that could be represented by a parametric equation in the TF plane.

Excision Hemorrhoidectomy: New Methods to Improve the Outcomes of an Old Technique

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Heng

2016-02-01

Full Text Available Context Hemorrhoidal disease is the most common anorectal disorder that requires surgical intervention. Hemorrhoids require treatment when they result in symptoms such as bleeding or prolapse. Surgical intervention is indicated for significant prolapse, and a number of accepted and viable methods are available for treating prolapsing hemorrhoids that do not reduce spontaneously (Grade III and IV. Excision hemorrhoidectomy remains the gold standard treatment for Grade III and IV hemorrhoids despite great interest in alternative procedures such as stapled hemorrhoidopexy and Doppler-guided hemorrhoidal artery ligation with mucopexy. A large body of evidence demonstrates that excision hemorrhoidectomy is an effective, safe, and affordable procedure. Nevertheless, the main drawback of excision hemorrhoidectomy remains its notorious association with significant postoperative pain. Evidence Acquisition A comprehensive literature search was conducted through MEDLINE and the Cochrane database of systematic reviews. Only prospective case-controlled studies, review articles, and meta-analyses were considered. Results Many strategies have been put forward in the literature to address the issue of pain after excision hemorrhoidectomy. These strategies can be broadly categorized into surgical techniques (e.g., LigaSure hemorrhoidectomy and pharmacological adjuncts (e.g., intradermal methylene blue and chemical sphincterotomy with glyceryl trinitrate ointment. In recent years, meta-analyses and randomized controlled trials have been performed to evaluate their effects. Conclusions This article evaluates the evidence behind these strategies and outlines the new methods available to improve the outcomes of an old technique.

DNA excision repair as a component of adaptation to low doses of ionizing radiation Escherichia coli

International Nuclear Information System (INIS)

Huang, H.; Claycamp, H.G.

1993-01-01

In this study the authors examined whether or not DNA excision repair is a component of adaptation induced by very low-dose ionizing radiation in Escherichia coli, a well-characterized prokaryote, and investigated the relationship between enhanced excision repair and the SOS response. Their data suggest that there seems to be narrow 'windows' of dose-effect for the induction of SOS-independent DNA excision repair. Being similar to mammalian cell studies, the dose range for this effect was about 200-fold less than D 37 for radiation survival. (author)

Does Excision of Heterotopic Ossification of the Elbow Result in Satisfactory Patient-Rated Outcomes?

Science.gov (United States)

Sandeep, K N; Suresh, G; Gopisankar, B; Abhishek, N; Sujiv, A

2017-03-01

Treatment of heterotopic ossification (HO) of the elbow is challenging and fraught with complications. Patients who sustain direct trauma to the elbow joint, the central nervous system, and thermal burns are at increased risk for development of HO. There is a paucity of studies and reports on patient's self-evaluation after the excision of the heterotopic ossification. This retrospective study assessed outcomes after excision of heterotopic ossification around the elbow in a cohort of ten patients operated from 2012 to 2015. The outcome assessment was done by the Mayo Elbow Performance index (MEPI) and the American Shoulder and Elbow Surgeons-Elbow score (ASES-E scores). The mean follow-up was 18.11 months after the operation. The Mayo Elbow Performance Score was excellent in two elbows, good in six and fair in two. The mean gain in flexion-extension arc after excision of HO was 80 degrees. All of the patients had residual flexion deformity postoperatively. Eight of the nine patients were able to do activities requiring flexion at final follow-up. Excision of HO around the elbow is associated with satisfactory patient-rated outcomes in spite of failure to regain full range of motion.

Isolated myocardial hydatid cyst: Managed with total curative excision

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Santosh Kumar Sinha

2016-01-01

Full Text Available Hydatid disease is still prevalent in developing countries, and isolated cardiac hydatid cysts are the rarest presentation. We report a 40-year-old nondiabetic, nonhypertensive female who presented with low-grade fever for 2 months shortness of breath and orthopnea for 2 weeks. Transthoracic echocardiography revealed a large, round cystic lesion with multiple daughter cysts without any obvious intraluminal detached membranes with mass effect on the left ventricular outflow tract. After total excision, residual tissue was closed with Teflon patch. Germinative membrane and hundreds of daughter cysts were seen. Following total excision of the cyst from myocardium, myocardial cavity was washed thoroughly with 10% Betadine solution. Pathological examination confirmed the diagnosis of hydatid cyst. Preoperatively started albendazole was continued for 4 weeks even after the operation. On follow-up after 4 weeks, the patient is doing well and cardiac imaging showed normal contours of the heart.

Excise Tax Avoidance: The Case of State Cigarette Taxes

Science.gov (United States)

DeCicca, Philip; Kenkel, Donald; Liu, Feng

2013-01-01

We conduct an applied welfare economics analysis of cigarette tax avoidance. We develop an extension of the standard formula for the optimal Pigouvian corrective tax to incorporate the possibility that consumers avoid the tax by making purchases in nearby lower-tax jurisdictions. To provide a key parameter for our formula, we estimate a structural endogenous switching regression model of border-crossing and cigarette prices. In illustrative calculations, we find that for many states, after taking into account tax avoidance the optimal tax is at least 20 percent smaller than the standard Pigouvian tax that simply internalizes external costs. Our empirical estimate that tax avoidance strongly responds to the price differential is the main reason for this result. We also use our results to examine the benefits of replacing avoidable state excise taxes with a harder-to-avoid federal excise tax on cigarettes. PMID:24140760

Excise tax avoidance: the case of state cigarette taxes.

Science.gov (United States)

DeCicca, Philip; Kenkel, Donald; Liu, Feng

2013-12-01

We conduct an applied welfare economics analysis of cigarette tax avoidance. We develop an extension of the standard formula for the optimal Pigouvian corrective tax to incorporate the possibility that consumers avoid the tax by making purchases in nearby lower tax jurisdictions. To provide a key parameter for our formula, we estimate a structural endogenous switching regression model of border-crossing and cigarette prices. In illustrative calculations, we find that for many states, after taking into account tax avoidance the optimal tax is at least 20% smaller than the standard Pigouvian tax that simply internalizes external costs. Our empirical estimate that tax avoidance strongly responds to the price differential is the main reason for this result. We also use our results to examine the benefits of replacing avoidable state excise taxes with a harder-to-avoid federal excise tax on cigarettes. Copyright © 2013 Elsevier B.V. All rights reserved.

Antibiotics for infection prevention after excision of the cervical transformation zone.

Science.gov (United States)

Kietpeerakool, Chumnan; Chumworathayi, Bandit; Thinkhamrop, Jadsada; Ussahgij, Butsakorn; Lumbiganon, Pisake

2017-01-21

Excision of the transformation zone of the cervix is the most commonly used approach to treat cervical precancerous lesions (cervical intraepithelial neoplasia (CIN)) to reduce the risk of developing cervical cancer. As the excision of the transformation zone leaves a raw area on the cervix, there is a risk of infection following the procedure. The incidence of infection after cold knife conization (CKC) is 36%, whereas the incidence for large loop excision of the transformation zone (LLETZ, also known as loop electrical excision procedure (LEEP)) is much lower (0.8% to 14.4%). Prophalytic antibiotics may prevent an infection developing and are often prescribed for CKC. However, there are no formal recommendations regarding the use of prophylactic antibiotics for infection prevention in women undergoing surgical excisional treatment for cervical precancerous lesions. To evaluate the effectiveness and safety of antibiotics for infection prevention following excision of the cervical transformation zone. We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (2016, Issue 4), MEDLINE, Embase, LILACS to May 2016. We also checked registers of clinical trials, citation lists of included studies, key textbooks and previous systematic reviews for potentially relevant studies SELECTION CRITERIA: We included randomised controlled trials (RCTs) evaluating the effectiveness and safety of prophylactic antibiotics versus a placebo or no treatment in women having excision of the cervical transformation zone, regardless of the type of surgical excisional method used. We used standard methodological procedures expected by Cochrane. Two review authors independently selected potentially relevant trials, extracted data, and assessed risk of bias, compared results and resolved disagreements by discussion. We contacted investigators for additional data, where possible. Of the 370 records that we identified as a result of the search (excluding duplicates), we regarded

Do alcohol excise taxes affect traffic accidents? Evidence from Estonia.

Science.gov (United States)

Saar, Indrek

2015-01-01

This article examines the association between alcohol excise tax rates and alcohol-related traffic accidents in Estonia. Monthly time series of traffic accidents involving drunken motor vehicle drivers from 1998 through 2013 were regressed on real average alcohol excise tax rates while controlling for changes in economic conditions and the traffic environment. Specifically, regression models with autoregressive integrated moving average (ARIMA) errors were estimated in order to deal with serial correlation in residuals. Counterfactual models were also estimated in order to check the robustness of the results, using the level of non-alcohol-related traffic accidents as a dependent variable. A statistically significant (P traffic accidents was disclosed under alternative model specifications. For instance, the regression model with ARIMA (0, 1, 1)(0, 1, 1) errors revealed that a 1-unit increase in the tax rate is associated with a 1.6% decrease in the level of accidents per 100,000 population involving drunk motor vehicle drivers. No similar association was found in the cases of counterfactual models for non-alcohol-related traffic accidents. This article indicates that the level of alcohol-related traffic accidents in Estonia has been affected by changes in real average alcohol excise taxes during the period 1998-2013. Therefore, in addition to other measures, the use of alcohol taxation is warranted as a policy instrument in tackling alcohol-related traffic accidents.

Inducible, tunable and multiplex human gene regulation using CRISPR-Cpf1-based transcription factors | Office of Cancer Genomics

Science.gov (United States)

Targeted and inducible regulation of mammalian gene expression is a broadly important research capability that may also enable development of novel therapeutics for treating human diseases. Here we demonstrate that a catalytically inactive RNA-guided CRISPR-Cpf1 nuclease fused to transcriptional activation domains can up-regulate endogenous human gene expression. We engineered drug-inducible Cpf1-based activators and show how this system can be used to tune the regulation of endogenous gene transcription in human cells.

Inverted 'V' osteotomy excision arthroplasty for bony ankylosed elbows

Directory of Open Access Journals (Sweden)

Rex Chadrabose

2011-12-01

Full Text Available Abstract Background Bony ankylosis of elbow is challenging and difficult problem to treat. The options are excision arthroplasty and total elbow replacement. We report our midterm results on nine patients, who underwent inverted 'V' osteotomy excision arthroplasty in our hospital with good functional results. Materials Our case series includes 9 patients (seven males and two females with the mean age of 34 years (13-56 years. Five patients had trauma, two had pyogenic arthritis, one had tuberculous arthritis, and one had pyogenic arthritis following surgical fixation. Results The average duration of follow up is 65 months (45 months-80 months. The mean Mayo's elbow performance score (MEPS preoperatively was 48 (35-70. The MEPS at final follow up was 80 (60-95. With no movement at elbow and fixed in various degrees of either flexion or extension preoperatively, the mean preoperative position of elbow was 64°(30°to 100°. The mean post operative range of motion at final follow up was 27°of extension (20-500, 116°of flexion (1100-1300, and the arc of motion was 88°(800-1000. One patient had ulnar nerve neuropraxia and another patient developed median nerve neuropraxia, and both recovered completely in six weeks. No patient had symptomatic instability of the elbow. All patients were asymptomatic except one patient, who had pain mainly on heavy activities. Conclusion We conclude that inverted 'V' osteotomy excision arthroplasty is a viable option in the treatment of bony ankylosis of the elbow in young patients.

Mutagenesis and phenotyping resources in zebrafish for studying development and human disease

Science.gov (United States)

Varshney, Gaurav Kumar

2014-01-01

The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts. PMID:24162064

Use of intracranial and ocular thermography before and after arteriovenous malformation excision

Science.gov (United States)

Hwang, Peter Y. K.; Lewis, Philip M.; Maller, Jerome J.

2014-11-01

Excision of arteriovenous malformations (AVMs) is known to carry a risk of postoperative hemorrhage, postulated to be the result of normal perfusion pressure breakthrough. It is also possible that AVMs may cause a steal effect, reducing perfusion in nearby vessels. There is currently no simple method of visualizing the presence or absence of steal effect intraoperatively. We hypothesized that the infrared thermographic (heat sensitive) imaging of perilesional brain may be useful for detecting reduced perfusion due to steal. Moreover, we hypothesized that if steal effect was present, it could impact on ocular perfusion and thereby temperature. Our objective was, therefore, to investigate whether perilesional cortical and ocular temperature (OT) may be a marker of steal effect. We intraoperatively acquired conventional and thermal images of the surgical field and eyes bilaterally, pre- and post-excisions of a large left hemisphere AVM. We found OT asymmetry preoperatively, which was absent after the AVM was excised. Intraoperative thermal images showed an increase of perilesional temperature, although this could be confounded by generalized changes in cortical perfusion due to anesthetics or surgery.

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

Science.gov (United States)

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

2015-01-01

To correct a DNA mutation in the human genome for gene therapy, homology-directed repair (HDR) needs to be specific and have the lowest off-target effects to protect the human genome from deleterious mutations. Zinc finger nucleases, transcription activator-like effector nuclease (TALEN) and CRISPR-CAS9 systems have been engineered and used extensively to recognize and modify specific DNA sequences. Although TALEN and CRISPR/CAS9 could induce high levels of HDR in human cells, their genotoxicity was significantly higher. Here, we report the creation of a monomeric endonuclease that can recognize at least 33 bp by fusing the DNA-recognizing domain of TALEN (TALE) to a re-engineered homing endonuclease I-SceI. After sequentially re-engineering I-SceI to recognize 18 bp of the human β-globin sequence, the re-engineered I-SceI induced HDR in human cells. When the re-engineered I-SceI was fused to TALE (TALE-ISVB2), the chimeric endonuclease induced the same HDR rate at the human β-globin gene locus as that induced by TALEN, but significantly reduced genotoxicity. We further demonstrated that TALE-ISVB2 specifically targeted at the β-globin sequence in human hematopoietic stem cells. Therefore, this monomeric endonuclease has the potential to be used in therapeutic gene targeting in human cells. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Nrf1 CNC-bZIP protein promotes cell survival and nucleotide excision repair through maintaining glutathione homeostasis.

Science.gov (United States)

Han, Weinong; Ming, Mei; Zhao, Rui; Pi, Jingbo; Wu, Chunli; He, Yu-Ying

2012-05-25

Skin cancer is the most common cancer in the United States. Its major environmental risk factor is UVB radiation in sunlight. In response to UVB damage, epidermal keratinocytes activate a specific repair pathway, i.e. nucleotide excision repair, to remove UVB-induced DNA lesions. However, the regulation of UVB response is not fully understood. Here we show that the long isoform of the nuclear factor erythroid 2-related factor 1 (Nrf1, also called NFE2L1), a cytoprotective transcription factor critical for the expression of multiple antioxidant response element-dependent genes, plays an important role in the response of keratinocytes to UVB. Nrf1 loss sensitized keratinocytes to UVB-induced apoptosis by up-regulating the expression of the proapoptotic Bcl-2 family member Bik through reducing glutathione levels. Knocking down Bik reduced UVB-induced apoptosis in Nrf1-inhibited cells. In UVB-irradiated surviving cells, however, disruption of Nrf1 impaired nucleotide excision repair through suppressing the transcription of xeroderma pigmentosum C (XPC), a factor essential for initiating the global genome nucleotide excision repair by recognizing the DNA lesion and recruiting downstream factors. Nrf1 enhanced XPC expression by increasing glutathione availability but was independent of the transcription repressor of XPC. Adding XPC or glutathione restored the DNA repair capacity in Nrf1-inhibited cells. Finally, we demonstrate that Nrf1 levels are significantly reduced by UVB radiation in mouse skin and are lower in human skin tumors than in normal skin. These results indicate a novel role of Nrf1 in UVB-induced DNA damage repair and suggest Nrf1 as a tumor suppressor in the skin.

Selective base excision repair of DNA damage by the non-base-flipping DNA glycosylase AlkC

Energy Technology Data Exchange (ETDEWEB)

Shi, Rongxin; Mullins, Elwood A.; Shen, Xing; #8208; Xing; Lay, Kori T.; Yuen, Philip K.; David, Sheila S.; Rokas, Antonis; Eichman, Brandt F. (UCD); (Vanderbilt)

2017-10-20

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.

Interference Excision in Spread Spectrum Communications Using Adaptive Positive Time-Frequency Analysis

Directory of Open Access Journals (Sweden)

Sridhar Krishnan

2007-07-01

Full Text Available This paper introduces a novel algorithm to excise single and multicomponent chirp-like interferences in direct sequence spread spectrum (DSSS communications. The excision algorithm consists of two stages: adaptive signal decomposition stage and directional element detection stage based on the Hough-Radon transform (HRT. Initially, the received spread spectrum signal is decomposed into its time-frequency (TF functions using an adaptive signal decomposition algorithm, and the resulting TF functions are mapped onto the TF plane. We then use a line detection algorithm based on the HRT that operates on the image of the TF plane and detects energy varying directional elements that satisfy a parametric constraint. Interference is modeled by reconstructing the corresponding TF functions detected by the HRT, and subtracted from the received signal. The proposed technique has two main advantages: (i it localizes the interferences on the TF plane with no cross-terms, thus facilitating simple filtering techniques based on thresholding of the TF functions, and is an efficient way to excise the interference; (ii it can be used for the detection of any directional interferences that can be parameterized. Simulation results with synthetic models have shown successful performance with linear and quadratic chirp interferences for single and multicomponent interference cases. The proposed method excises the interference even under very low SNR conditions of −10 dB, and the technique could be easily extended to any interferences that could be represented by a parametric equation in the TF plane.

Functional evaluation of DNA repair in human biopsies and their relation to other cellular biomarkers

Czech Academy of Sciences Publication Activity Database

Slyšková, Jana; Langie, S. A. S.; Collins, A. R.; Vodička, Pavel

2014-01-01

Roč. 116, č. 5 (2014) ISSN 1664-8021 R&D Projects: GA ČR(CZ) GAP304/12/1585 Institutional support: RVO:68378041 Keywords : base excision repair * nucleotide excision repair * human solid tissue Subject RIV: EB - Genetics ; Molecular Biology

The safety of complete mesocolic excision once again confirmed

DEFF Research Database (Denmark)

Bertelsen, C A; Neuenschwander, A U

2018-01-01

We would like to commend Bernhoff and colleagues (1) for publishing further evidence supporting complete mesocolic excision (CME) as a safe approach in colon cancer surgery. They report no increased risk of 30-day mortality or reoperation after right-sided CME in a nested case-control study...

Clinical trial comparing excision and primary closure with modified ...

African Journals Online (AJOL)

The aim of this study was to compare modified Limberg flap procedure with excision and primary closure in the treatment of uncomplicated pilonidal disease. Methods: This study was conducted on 120 patients with uncomplicated sacrococcygeal pilonidal disease that were randomly allocated into two groups: group I ...

Deficiency of gamma-ray excision repair in skin fibroblasts from patients with Fanconi's anemia

International Nuclear Information System (INIS)

Remsen, J.F.; Cerutti, P.A.

1976-01-01

The capacity of preparations of skin fibroblasts from normal individuals and patients with Fanconi's anemia to excise gamma-ray products of the 5,6-dihydroxydihydrothymine type from exogenous DNA was investigated. The excision capacity of whole-cell homogenates of fibroblasts from two of four patients with Fanconi's anemia was substantially below normal. This repair deficiency was further pronounced in nuclear preparations from cells of the same two patients

Human inherited diseases with altered mechanisms for DNA repair and mutagenesis

Energy Technology Data Exchange (ETDEWEB)

Cleaver, J.E.

1977-01-01

A variety of human diseases involving clinical symptoms of increased cancer risk, and disorders of the central nervous system, and of hematopoietic, immunological, ocular, and cutaneous tissues and embryological development have defects in biochemical pathways for excision repair of damaged DNA. Excision repair has multiple branches by which damaged nucleotides, bases, and cross-links are excised and requires cofactors that control the access of repair enzymes to damage in DNA in chromatin. Diseases in which repair defects are a consistent feature of their biochemistry include xeroderma pigmentosum, ataxia telangiectasia and Fanconi's anemia.

Pilonidal sinus disease surgery in children: the first study to compare crystallized phenol application to primary excision and closure.

Science.gov (United States)

Ates, Ufuk; Ergun, Ergun; Gollu, Gulnur; Sozduyar, Sumeyye; Kologlu, Meltem; Cakmak, Murat; Dindar, Huseyin; Yagmurlu, Aydin

2018-03-01

Pilonidal sinus (PS) is an infectious and inflammatory disease of sacrococcygeal region. Current methods include; surgical excision with/without suturing the defect, rhomboid excision and flap and chemical substance application. In this study, crystallized phenol application was compared to excision and primary closure. This retrospective study included pediatric patients with PS who were treated with excision and primer closure technique and phenol application. The patients' medical data were analyzed retrospectively. This study included 117 patients with PS. There were 52 girls (44%) and 65 boys (56%). Mean age of children was 15.6 (12-20) years. Excision and primary closure were applied to 77 patients (66%) and phenol was applied to 40 patients (34%). The children in phenol group were discharged on the operation day; mean hospitalization time in the excision and primary closure group was 2.7 (1-14) days. Mean follow up was 44.6 (8-82) months for primary excision and closure group and 8.1 (1-19) months for phenol group. Although many surgical and non-surgical treatment modalities have been described for PS, the optimal one remains unknown. Limited with the retrospective nature of the data, crystallized phenol application seems a feasible minimal invasive alternative to primary closure of PS with lower recurrence and complication rates in children. Level III. Copyright © 2017 Elsevier Inc. All rights reserved.

26 CFR 55.4982-1 - Imposition of excise tax on undistributed income of regulated investment companies.

Science.gov (United States)

2010-04-01

... 26 Internal Revenue 17 2010-04-01 2010-04-01 false Imposition of excise tax on undistributed income of regulated investment companies. 55.4982-1 Section 55.4982-1 Internal Revenue INTERNAL REVENUE... ESTATE INVESTMENT TRUSTS AND REGULATED INVESTMENT COMPANIES Excise Tax on Regulated Investment Companies...

Post-operative outcomes following the excision of dorsal wrist ganglions with/without the use of Methylene Blue

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Suleyman Tas

2016-04-01

Conclusions: The recurrence of cysts is rare after complete excision, particularly when rupture is prevented. Methylene blue injection is useful for ensuring complete excision and, therefore, to prevent recurrence. [Hand Microsurg 2016; 5(1.000: 1-6

A human homolog of the yeast nucleotide excision repair gene MMS19 interacts with transcription repair factor TFIIH through the XPB and XPD helicases.

NARCIS (Netherlands)

T. Seroz; G.S. Winkler (Sebastiaan); J. Auriol; R.A. Verhage; W. Vermeulen (Wim); B. Smit (Bep); J. Brouwer (Jaap); A.P.M. Eker (André); G. Weeda (Geert); J-M. Egly (Jean-Marc); J.H.J. Hoeijmakers (Jan)

2000-01-01

textabstractNucleotide excision repair (NER) removes UV-induced photoproducts and numerous other DNA lesions in a highly conserved 'cut-and-paste' reaction that involves approximately 25 core components. In addition, several other proteins have been identified which are dispensable for NER in vitro

Surgical Approaches to First Branchial Cleft Anomaly Excision: A Case Series

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Lourdes Quintanilla-Dieck

2016-01-01

Full Text Available Objectives. First branchial cleft anomalies (BCAs constitute a rare entity with variable clinical presentations and anatomic findings. Given the high rate of recurrence with incomplete excision, identification of the entire tract during surgical treatment is of paramount importance. The objectives of this paper were to present five anatomic variations of first BCAs and describe the presentation, evaluation, and surgical approach to each one. Methods. A retrospective case review and literature review were performed. We describe patient characteristics, presentation, evaluation, and surgical approach of five patients with first BCAs. Results. Age at definitive surgical treatment ranged from 8 months to 7 years. Various clinical presentations were encountered, some of which were atypical for first BCAs. All had preoperative imaging demonstrating the tract. Four surgical approaches required a superficial parotidectomy with identification of the facial nerve, one of which revealed an aberrant facial nerve. In one case the tract was found to travel into the angle of the mandible, terminating as a mandibular cyst. This required en bloc excision that included the lateral cortex of the mandible. Conclusions. First BCAs have variable presentations. Complete surgical excision can be challenging. Therefore, careful preoperative planning and the recognition of atypical variants during surgery are essential.

Surgical Approaches to First Branchial Cleft Anomaly Excision: A Case Series.

Science.gov (United States)

Quintanilla-Dieck, Lourdes; Virgin, Frank; Wootten, Chistopher; Goudy, Steven; Penn, Edward

2016-01-01

Objectives. First branchial cleft anomalies (BCAs) constitute a rare entity with variable clinical presentations and anatomic findings. Given the high rate of recurrence with incomplete excision, identification of the entire tract during surgical treatment is of paramount importance. The objectives of this paper were to present five anatomic variations of first BCAs and describe the presentation, evaluation, and surgical approach to each one. Methods. A retrospective case review and literature review were performed. We describe patient characteristics, presentation, evaluation, and surgical approach of five patients with first BCAs. Results. Age at definitive surgical treatment ranged from 8 months to 7 years. Various clinical presentations were encountered, some of which were atypical for first BCAs. All had preoperative imaging demonstrating the tract. Four surgical approaches required a superficial parotidectomy with identification of the facial nerve, one of which revealed an aberrant facial nerve. In one case the tract was found to travel into the angle of the mandible, terminating as a mandibular cyst. This required en bloc excision that included the lateral cortex of the mandible. Conclusions. First BCAs have variable presentations. Complete surgical excision can be challenging. Therefore, careful preoperative planning and the recognition of atypical variants during surgery are essential.

Osteochondroma of the mandibular condyle: surgical excision followed by occlusal stabilization.

Science.gov (United States)

Yoo, Ji-Yeon; Choi, Byung-Joon; Kwon, Yong-Dae; Lee, Deok-Won; Ohe, Joo-Young; Suh, Joon-Ho

2012-03-01

Osteochondroma of the mandibular condyle in adults can be treated by surgical excision, condylectomy followed by costochondral graft or orthognathic surgery. Such complex treatment plan may not be appropriate for patients with old age, affected with chronic osteochondroma of the condyle. In this clinical report, we present a patient with osteochondroma of the condyle treated by surgical excision. The patient's postoperative occlusion was a contraindication for orthognathic surgery because of the severe abrasion of the teeth and the chronic compensation of the dentition to the deviated mandible. Surgical excision of the lesion was carried out under general anesthesia, and the remaining condylar head was salvaged as much as possible. No graft materials or posthodontic condyle reconstruction was carried out. Because there was no occlusal stop to secure the mandible in a centric relation position of the condyle, a stabilization splint was delivered to position the condyle in a relatively stable position. The stability of the condyle position was evaluated by follow-up cone beam computed tomographic scans of the pathologic and the contralateral condyle, along with clinical factors such as occlusal contact points and mandible movements assayed by ARCUSdigma (KaVo). After significant condylar position was achieved, full prosthodontic reconstruction was performed to both the patient's and the dentist's satisfaction.

A new approach to laparoscopic lymph node excision in cases of transverse colon cancer.

Science.gov (United States)

Nakanishi, Masayoshi; Kokuba, Yukihito; Murayama, Yasutoshi; Komatsu, Shuhei; Shiozaki, Atsushi; Kuriu, Yoshiaki; Ikoma, Hisashi; Ichikawa, Daisuke; Fujiwara, Hitoshi; Okamoto, Kazuma; Ochiai, Toshiya; Otsuji, Eigo

2012-01-01

Treatment of transverse colon cancer by laparoscopic surgery is difficult, and this surgery has been excluded in many randomized control trials. Difficulty in excising lymph nodes around the middle colonic artery has been the main factor responsible for the complexity of this surgery. Herein, we describe a new approach to overcome this difficulty in lymph node excision in cases of transverse colon cancer. We adopted the following steps to collect information that was otherwise difficult to obtain from two-dimensional images displayed on the monitor screen, in order to ensure safety during laparoscopic surgery. (1) The omental bursa was opened by directly visualizing it through a small incision created in the median epigastric region, and the cranial side of the transverse colon mesentery was then freed. (2) The colonic drainage vein entering the right gastroepiploic vein was dissected, and a gauze was inserted into the freed layer. (3) Under laparoscopic guidance, the freed layer was fixed, with the inserted gauze serving as a landmark. The lymph nodes were then excised making full use of the horizontal view. Utilization of a small incision in the abdomen enables full use of the horizontal view for manipulations during laparoscopy, allowing safe manipulations for lymph node excision. Copyright © 2012 S. Karger AG, Basel.

Trans-sphenoidal excision of craniopharyngiomas.

Directory of Open Access Journals (Sweden)

Nagpal R

1991-04-01

Full Text Available Craniopharyngiomas have been by and large excised by the transcranial route. Since 1982, 11 patients have been operated by the traus-sphensidal route in the department. The clinical features with which they were presented, diagnostic investigations, details of surgical procedures and follow-up analysis is being presented here. A retrospective study of radiological investigation was done to determine the features that help decide the choice of surgical approach to these lesions. Only lesions that were primarily intrasellar, cystic and those that expanded the sella could be treated by the trans-sphenoidal route. Associated suprasellar extensions could also be removed. Predominantly calcified or firm, fleshy tumours lent themselves poorly to removal by the trans-sphenoidal route.

Capacity of ultraviolet-induced DNA repair in human glioma cells

Energy Technology Data Exchange (ETDEWEB)

Itoh, Hiroji

1987-04-01

A DNA repair abnormality is likely related to an increased incidence of neoplasms in several autosomal recessive diseases such as xeroderma pigmentosum, Fanconi's anemia, Bloom's syndrome and ataxia telangiectasia. In human glioma cells, however, there are only a few reports on DNA repair. In this study, an ultraviolet (UV)-induced DNA repair was examined systematically in many human glioma cells. Two human malignant glioma cell lines (MMG-851, U-251-MG) and 7 human glioma cell strains (4, benign; 3, malignant) of short term culture, in which glial fibrillary acidic protein (GFAP) staining were positive, were used. To investigate the capacity of DNA repair, UV sensitivity was determined by colony formation; excision repair by autoradiography and Cytosine Arabinoside (Ara-C) assay; and post-replication repair by the joining rate of newly synthesized DNA. As a result, the colony-forming abilities of malignant glioma cell lines were lower than those of normal human fibroblasts, but no difference was found between two malignant glioma cell lines. The excision repair of the malignant group (2 cell lines and 3 cell strains) was apparently lower than that of the benign group (4 cell strains). In two malignant glioma cell lines, the excision repair of MMG-851 was lower than that of U-251-MG, and the post-replication repair of MMG-851 was higher than that of U-251-MG. These results were considered to correspond well with colony-forming ability. The results indicate that there are some differences in each human malignant glioma cell in its UV-induced DNA repair mechanism, and that the excision repair of the malignant glioma cells is apparently lower than that of the benign glioma cells. These findings may be useful for diagnosis and treatment.

Surgical excision of lung metastases from squamous carcinoma of ...

African Journals Online (AJOL)

These 2 case reports serveto emphasizetwo important points concerning carcinoma of the cervix: (i) blood-borne metastases are now frequently encountered in this disease; and (ii). in selected cases surgical excision of a secondary deposit in the lung is the treatment of choice and may even result -in cure.

Gene repair of an Usher syndrome causing mutation by zinc-finger nuclease mediated homologous recombination.

Science.gov (United States)

Overlack, Nora; Goldmann, Tobias; Wolfrum, Uwe; Nagel-Wolfrum, Kerstin

2012-06-26

Human Usher syndrome (USH) is the most frequent cause of inherited deaf-blindness. It is clinically and genetically heterogeneous, assigned to three clinical types of which the most severe type is USH1. No effective treatment for the ophthalmic component of USH exists. Gene augmentation is an attractive strategy for hereditary retinal diseases. However, several USH genes, like USH1C, are expressed in various isoforms, hampering gene augmentation. As an alternative treatment strategy, we applied the zinc-finger nuclease (ZFN) technology for targeted gene repair of an USH1C, causing mutation by homologous recombination. We designed ZFNs customized for the p.R31X nonsense mutation in Ush1c. We evaluated ZFNs for DNA cleavage capability and analyzed ZFNs biocompatibilities by XTT assays. We demonstrated ZFNs mediated gene repair on genomic level by digestion assays and DNA sequencing, and on protein level by indirect immunofluorescence and Western blot analyses. The specifically designed ZFNs did not show cytotoxic effects in a p.R31X cell line. We demonstrated that ZFN induced cleavage of their target sequence. We showed that simultaneous application of ZFN and rescue DNA induced gene repair of the disease-causing mutation on the genomic level, resulting in recovery of protein expression. In our present study, we analyzed for the first time ZFN-activated gene repair of an USH gene. The data highlight the ability of ZFNs to induce targeted homologous recombination and mediate gene repair in USH. We provide further evidence that the ZFN technology holds great potential to recover disease-causing mutations in inherited retinal disorders.

2-cm versus 4-cm surgical excision margins for primary cutaneous melanoma thicker than 2 mm: a randomised, multicentre trial

DEFF Research Database (Denmark)

Gillgren, Peter; Drzewiecki, Krzysztof T; Niin, Marianne

2011-01-01

Optimum surgical resection margins for patients with clinical stage IIA-C cutaneous melanoma thicker than 2 mm are controversial. The aim of the study was to test whether survival was different for a wide local excision margin of 2 cm compared with a 4-cm excision margin.......Optimum surgical resection margins for patients with clinical stage IIA-C cutaneous melanoma thicker than 2 mm are controversial. The aim of the study was to test whether survival was different for a wide local excision margin of 2 cm compared with a 4-cm excision margin....

Efficient CRISPR/Cas9-Based Genome Engineering in Human Pluripotent Stem Cells.

Science.gov (United States)

Kime, Cody; Mandegar, Mohammad A; Srivastava, Deepak; Yamanaka, Shinya; Conklin, Bruce R; Rand, Tim A

2016-01-01

Human pluripotent stem cells (hPS cells) are rapidly emerging as a powerful tool for biomedical discovery. The advent of human induced pluripotent stem cells (hiPS cells) with human embryonic stem (hES)-cell-like properties has led to hPS cells with disease-specific genetic backgrounds for in vitro disease modeling and drug discovery as well as mechanistic and developmental studies. To fully realize this potential, it will be necessary to modify the genome of hPS cells with precision and flexibility. Pioneering experiments utilizing site-specific double-strand break (DSB)-mediated genome engineering tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have paved the way to genome engineering in previously recalcitrant systems such as hPS cells. However, these methods are technically cumbersome and require significant expertise, which has limited adoption. A major recent advance involving the clustered regularly interspaced short palindromic repeats (CRISPR) endonuclease has dramatically simplified the effort required for genome engineering and will likely be adopted widely as the most rapid and flexible system for genome editing in hPS cells. In this unit, we describe commonly practiced methods for CRISPR endonuclease genomic editing of hPS cells into cell lines containing genomes altered by insertion/deletion (indel) mutagenesis or insertion of recombinant genomic DNA. Copyright © 2016 John Wiley & Sons, Inc.

Management of post-traumatic elbow instability after failed radial head excision: A case report

OpenAIRE

Touloupakis, Georgios; Theodorakis, Emmanouil; Favetti, Fabio; Nannerini, Massimiliano

2017-01-01

Radial head excision has always been a safe commonly used surgical procedure with a satisfactory clinical outcome for isolated comminuted radial head fractures. However, diagnosis of elbow instability is still very challenging and often underestimated in routine orthopaedic evaluation. We present the case of a 21-years old female treated with excision after radial head fracture, resulting in elbow instability. The patient underwent revision surgery after four weeks. We believe that ligament r...

Induction of the Tn10 Precise Excision in E. coli Cells after Accelerated Heavy Ions Irradiation

CERN Document Server

Zhuravel, D V

2003-01-01

The influence of the irradiation of different kinds on the indication of the structural mutations in the bacteria Escherichia coli is considered. The regularities of the Tn10 precise excision after accelerated ^{4}He and ^{12}C ions irradiations with different linear energy transfer (LET) were investigated. Dose dependences of the survival and relative frequency of the Tn10 precise excision were obtained. It was shown, that the relative frequency of the Tn10 precise excision is the exponential function from the irradiation dose. Relative biological efficiency (RBE), and relative genetic efficiency (RGE) were calculated, and were treated as the function of the LET.

Repair of 3-methyladenine and abasic sites by base excision repair mediates glioblastoma resistance to temozolomide

Energy Technology Data Exchange (ETDEWEB)

Bobola, Michael S.; Kolstoe, Douglas D.; Blank, A. [Department of Neurological Surgery, University of Washington Medical Center, Seattle, WA (United States); Chamberlain, Marc C. [Department of Neurological Surgery, University of Washington Medical Center, Seattle, WA (United States); Department of Neurology, University of Washington Medical Center, Seattle, WA (United States); Silber, John R., E-mail: jrsilber@u.washington.edu [Department of Neurological Surgery, University of Washington Medical Center, Seattle, WA (United States)

2012-11-30

Alkylating agents have long played a central role in the adjuvant therapy of glioblastoma (GBM). More recently, inclusion of temozolomide (TMZ), an orally administered methylating agent with low systemic toxicity, during and after radiotherapy has markedly improved survival. Extensive in vitro and in vivo evidence has shown that TMZ-induced O{sup 6}-methylguanine (O{sup 6}-meG) mediates GBM cell killing. Moreover, low or absent expression of O{sup 6}-methylguanine-DNA methyltransferase (MGMT), the sole human repair protein that removes O{sup 6}-meG from DNA, is frequently associated with longer survival in GBMs treated with TMZ, promoting interest in developing inhibitors of MGMT to counter resistance. However, the clinical efficacy of TMZ is unlikely to be due solely to O{sup 6}-meG, as the agent produces approximately a dozen additional DNA adducts, including cytotoxic N3-methyladenine (3-meA) and abasic sites. Repair of 3-meA and abasic sites, both of which are produced in greater abundance than O{sup 6}-meG, is mediated by the base excision repair (BER) pathway, and occurs independently of removal of O{sup 6}-meG. These observations indicate that BER activities are also potential targets for strategies to potentiate TMZ cytotoxicity. Here we review the evidence that 3-meA and abasic sites mediate killing of GBM cells. We also present in vitro and in vivo evidence that alkyladenine-DNA glycosylase, the sole repair activity that excises 3-meA from DNA, and Ape1, the major human abasic site endonuclease, mediate TMZ resistance in GBMs and represent potential anti-resistance targets.

Excision versus trichloroacetic acid (TCA) chemocauterization for branchial sinus of the pyriform fossa.

Science.gov (United States)

Hwang, Jihee; Kim, Seong Chul; Kim, Dae Yeon; Namgoong, Jung-Man; Nam, Soon Yuhl; Roh, Jong-Lyel

2015-11-01

We analyzed the outcomes of open surgical excision and endoscopic trichloroacetic acid (TCA) chemocauterization for the treatment of branchial sinus of the pyriform fossa (BSPF). We retrospectively reviewed the records of 27 patients (16 males and 11 females) who were treated for BSPF at the Asan Medical Center between 1996 and 2013. The median age of the 27 patients was 4.5years (range, 0 to 15years). Before definitive surgery, 19 (70.3%) of the patients had histories of neck infection, and 16 (59.2%) patients had neck abscesses that were drained. The lesions were predominantly located on the left side (26 of 27; 96.2%). Excisions were performed for 14 (48.1%) patients. TCA chemocauterizations were performed for 13 patients. After a median follow-up period of 5.5years, 11 patients developed recurrence. The recurrence rates were not significantly different between the excision and chemocauterization groups (35.7% vs 46.1%, respectively, p=0.704). All of the recurred patients were successfully treated with repeated chemocauterization or reexcision. Analyses of the risk factors for recurrence revealed that a previous infection history tended to increase the rate of recurrence (90.9% vs 56.2%, p=0.090). Our experience suggests that the outcomes of excision and TCA chemocauterization are not significantly different. Additional studies are needed to reach a consensus regarding the best treatment strategy for BSPF. Copyright © 2015 Elsevier Inc. All rights reserved.

An ameliorative protocol for the quantification of purine 5',8-cyclo-2'-deoxynucleosides in oxidized DNA

Science.gov (United States)

Terzidis, Michael; Chatgilialoglu, Chryssostomos

2015-07-01

5',8-Cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG) are lesions resulting from hydroxyl radical (HO•) attack on the 5'H of the nucleoside sugar moiety and exist in both 5'R and 5'S diastereomeric forms. Increased levels of cdA and cdG are linked to Nucleotide Excision Repair mechanism deficiency and mutagenesis. Discrepancies in the damage measurements reported over recent years indicated the weakness of the actual protocols, in particular for ensuring the quantitative release of these lesions from the DNA sample and the appropriate method for their analysis. Herein we report the detailed revision leading to a cost-effective and efficient protocol for the DNA damage measurement, consisting of the nuclease benzonase and nuclease P1 enzymatic combination for DNA digestion followed by liquid chromatography isotope dilution tandem mass spectrometry analysis.

26 CFR 25.2512-7 - Effect of excise tax.

Science.gov (United States)

2010-04-01

... by a taxpayer and made the subject of gifts within a reasonable time after purchase, the purchase...-7 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) ESTATE AND GIFT TAXES GIFT TAX; GIFTS MADE AFTER DECEMBER 31, 1954 Transfers § 25.2512-7 Effect of excise tax. If...

Revenue and Health Impacts of Restructuring Tobacco Excise Tax ...

International Development Research Centre (IDRC) Digital Library (Canada)

A proposed law in the Philippines to increase the excise tax on tobacco by ... developing countries to reduce demand for tobacco through price and tax measures. Southeast Asian countries are struggling with how to implement the main provisions of the Convention. ... Far East Asia, Philippines, Central Asia, South Asia ...

The Role of Hemosiderin Excision in Seizure Outcome in Cerebral Cavernous Malformation Surgery: A Systematic Review and Meta-Analysis.

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Di Ruan

Full Text Available Whether the excision of hemosiderin surrounding cerebral cavernous malformations (CCMs is necessary to achieve a seizure-free result has been the subject of debate. Here, we report a systematic review of related literature up to Jan 1, 2015 including 594 patients to assess the effect of hemosiderin excision on seizure outcome in patients with CCMs by meta-analysis.Ten studies comparing extended hemosiderin excision with only lesion resection were identified by searching the English-language literature. Meta-analyses, subgroup analyses and sensitivity analysis were conducted to determine the association between hemosiderin excision and seizure outcome after surgery.Seizure outcome was significantly improved in the patients who underwent an extended excision of the surrounding hemosiderin (OR, 0.62; 95% CI: 0.42-0.91; P = 0.01. In subgroup analysis, studies from Asia (OR, 0.42; 95% CI: 0.25-0.71; P = 0.001, male-majority (female ratio 1 year before surgery (OR, 0.43; 95% CI: 0.22-0.84; P = 0.01, lesion diameter > 2 cm (OR, 0.41; 95% CI: 0.19-0.87; P = 0.02 and short-term (< 3 years follow-up (OR, 0.48; 95% CI: 0.29-0.80; P = 0.005 tended to correlate with a significantly favorable outcome.Patients who underwent extended surrounding hemosiderin excision could exhibit significantly improved seizure outcomes compared to patients without hemosiderin excision. However, further well-designed prospective multiple-center RCT studies are still needed.

Survival response of RIF tumor cells to heat-x-radiation combinations: Parallel measurements in culture and by the excision assay

International Nuclear Information System (INIS)

Henle, K.J.; Nagle, W.A.; Moss, A.J.

1984-01-01

The cytotoxicity of heat-radiation (hX) combinations in vivo may differ from that measured in vitro. The authors have used the RIF tumor, grown in mouse feet, to compare the survival response after in situ hX-treatments with identical hX in vitro. The radiation survival curve, determined by the excision assay showed a slightly larger D/sub o/ than that measured in vitro (250, 200 rad, respective) and survival measurements appeared independent of excision time after irradiation. The 45 0 -heat survival curve was similar in both assays, but only when the excision followed immediately after h. A 24-hr delayed excision removed the shoulder and lowered survival 30-fold after either 20 or 30 min, 45 0 . Similar survival values were measured after 10 min, 45 0 +X (hX) in vitro and with immediate excision, although the excision survival curve had no shoulder and a D/sub o/ of 180 rad vs. 120 rad in vitro. The survival curve with delayed excision (24 hr) also appeared as a simple exponential curve with an apparent D/sub o/ of 310 rad (n=0.02). Two fractions of combined hX, separated by 24 hr (hx+24+hX), yielded D/sub o/=90 rad, D/sub q/=230 rad in vitro but 370 and 400 rad, respectively, when measured by delayed excision. The apparent radioresistance in vivo is consistent with data by Song of increased hypoxic fractions after heating in vivo and argues against combining hx in every fraction for optimal tumor control

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

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Xinxia Zhao

2016-03-01

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

Evidence that DNA excision-repair in xeroderma pigmentosum group A is limited but biologically significant

International Nuclear Information System (INIS)

Hull, D.R.; Kantor, G.J.

1983-01-01

The loss of pyrimidine dimers in nondividing populations of an excision-repair deficient xeroderma pigmentosum group. A strain (XP12BE) was measured throughout long periods (up to 5 months) following exposure to low doses of ultraviolet light (UV, 254 nm) using a UV endonuclease-alkaline sedimentation assay. Excision of about 90% of the dimers induced by 1 J/m 2 occurred during the first 50 days. The rate curve has some similarities with that of normal excision-repair proficient cultures that may not be coincidental. Rate curves for both XP12BE and normal cultures are characterized by a fast and slow component, with both rate constants for the XP12BE cultures (0.15 day -1 and 0.025 day -1 ) a factor of 10 smaller than those observed for the respective components of normal cell cultures. The slow components for both XP12BE and normal cultures extrapolate to about 30% of the initial number of dimers. No further excision was detected throughout an additional 90-day period even though the cultures were capable of excision-repair of other newly-introduced pyrimidine dimers. We conclude that nondividing XP12BE cells in addition to having a slower repair rate, cannot repair some of the UV-induced DNA damage. The repair in XP12BE is shown to have biological significance as detected by a cell-survival assay and dose-fractionation techniques. Nondividing XP12BE cells are more resistant to UV when irradiated chronically than when irradiated acutely with the same total dose. (orig.)

High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

Science.gov (United States)

Bi, Yanwei; Sun, Le; Gao, Dandan; Ding, Chen; Li, Zhihua; Li, Yadong; Cun, Wei; Li, Qihan

2014-05-01

A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)) RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

Directory of Open Access Journals (Sweden)

Yanwei Bi

2014-05-01

Full Text Available A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR-associated (Cas RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ and homology-directed repair (HDR pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

Basal Cell Carcinoma of the Head and Neck Region: A Retrospective Analysis of Completely Excised 331 Cases

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Duriye Deniz Demirseren

2014-01-01

Full Text Available The aim of the study is to analyze all completely excised BCCs in the head and neck region with regard to age, sex, personal and familial history, skin type, tumor localization and size, histopathological subtype of tumor, reconstruction method, and recurrence rates. Incompletely excised BCCs were not included in this study since incomplete excision is the most important preventable risk factor for recurrence. In 320 patients, 331 lesions were retrospectively evaluated by dividing into the following 8 subunits: scalp, frontotemporal, orbital, nose, cheek, auricula, perioral, and chin-neck area. Most of the patients were in 60–70 age group (34.7%. The nose (32.3% was the most common site of presentation. Clinically, all lesions and, histopathologically, most of the lesions (42.2% presented were of the nodular type. All cases of recurrence after complete excision (n=9, 2.7% were located in the median parts of the head and neck region and were mainly diagnosed histopathologically as sclerotic and micronodular. Even though completely excised, head and neck region BCCs, especially which are more prone to recurrence due to anatomical and histopathological properties, should be more closely monitored in order to decrease morbidity and health care costs.

Quantitative imaging of excised osteoarthritic cartilage using spectral CT

Energy Technology Data Exchange (ETDEWEB)

Rajendran, Kishore; Bateman, Christopher J.; Younis, Raja Aamir; De Ruiter, Niels J.A.; Ramyar, Mohsen; Anderson, Nigel G. [University of Otago - Christchurch, Department of Radiology, Christchurch (New Zealand); Loebker, Caroline [University of Otago, Christchurch Regenerative Medicine and Tissue Engineering Group, Department of Orthopaedic Surgery and Musculoskeletal Medicine, Christchurch (New Zealand); University of Twente, Department of Developmental BioEngineering, Enschede (Netherlands); Schon, Benjamin S.; Hooper, Gary J.; Woodfield, Tim B.F. [University of Otago, Christchurch Regenerative Medicine and Tissue Engineering Group, Department of Orthopaedic Surgery and Musculoskeletal Medicine, Christchurch (New Zealand); Chernoglazov, Alex I. [University of Canterbury, Human Interface Technology Laboratory New Zealand, Christchurch (New Zealand); Butler, Anthony P.H. [University of Otago - Christchurch, Department of Radiology, Christchurch (New Zealand); European Organisation for Nuclear Research (CERN), Geneva (Switzerland); MARS Bioimaging, Christchurch (New Zealand)

2017-01-15

To quantify iodine uptake in articular cartilage as a marker of glycosaminoglycan (GAG) content using multi-energy spectral CT. We incubated a 25-mm strip of excised osteoarthritic human tibial plateau in 50 % ionic iodine contrast and imaged it using a small-animal spectral scanner with a cadmium telluride photon-processing detector to quantify the iodine through the thickness of the articular cartilage. We imaged both spectroscopic phantoms and osteoarthritic tibial plateau samples. The iodine distribution as an inverse marker of GAG content was presented in the form of 2D and 3D images after applying a basis material decomposition technique to separate iodine in cartilage from bone. We compared this result with a histological section stained for GAG. The iodine in cartilage could be distinguished from subchondral bone and quantified using multi-energy CT. The articular cartilage showed variation in iodine concentration throughout its thickness which appeared to be inversely related to GAG distribution observed in histological sections. Multi-energy CT can quantify ionic iodine contrast (as a marker of GAG content) within articular cartilage and distinguish it from bone by exploiting the energy-specific attenuation profiles of the associated materials. (orig.)

Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.

Science.gov (United States)

Gao, Difeng; Smith, Spencer; Spagnuolo, Michael; Rodriguez, Gabriel; Blenner, Mark

2018-05-29

CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Keratoacanthoma centrifugum marginatum: unresponsive to oral retinoid and successfully treated with wide local excision.

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Kapildev Das

2010-01-01

Full Text Available We describe a case of a 65-year-old male presenting with a large plaque with a rolled-out interrupted margin, atrophic center, and island of normal skin over the left arm. It grew peripherally with central healing, and there was a history of recurrence after inadequate excision. Investigations ruled out other clin­ical mimickers; namely, squamous cell carcinoma, lupus vulgaris, botryomycosis, and blastomycosis-like pyoderma. Histopathological sections showed irregularly shaped craters filled with keratin and epithelial pearl but no evidence of granuloma or cellular atypia. Clinico­pathological correlation proved the lesion to be keratoacanthoma centrifugum marginatum (KCM, a rare variant of keratoacanthoma, which spreads centrifugally, attains a huge size, and never involutes spontaneously. Treatment of KCM has been a problem always and, in our case, systemic retinoid (acitretin for three months proved ineffective. The patient also had a history of recurrence following surgical intervention previously, necessitating wide excision to achieve complete clearance of tumor cells. Hence, after failure of retinoid therapy, the decision of excision with a 1-centimeter margin was taken and the large defect was closed by a split thickness skin graft. The graft uptake was satisfactory, and the patient is being followed-up presently and shows no signs of recurrence after six months, highlighting wide local excision as a useful treatment option.

Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance

Science.gov (United States)

Shahbazi Dastjerdeh, Mansoureh; Kouhpayeh, Shirin; Sabzehei, Faezeh; Khanahmad, Hossein; Salehi, Mansour; Mohammadi, Zahra; Shariati, Laleh; Hejazi, Zahra; Rabiei, Parisa; Manian, Mostafa

2016-01-01

Background: The evolution of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) has been accelerated recently by the indiscriminate application of antibiotics. Antibiotic resistance has challenged the success of medical interventions and therefore is considered a hazardous threat to human health. Objectives: The present study aimed to describe the use of zinc finger nuclease (ZFN) technology to target and disrupt a plasmid-encoded β-lactamase, which prevents horizontal gene transfer-mediated evolution of ARBs. Materials and Methods: An engineered ZFN was designed to target a specific sequence in the ampicillin resistance gene (ampR) of the pTZ57R plasmid. The Escherichia coli bacteria already contained the pZFN kanamycin-resistant (kanaR) plasmid as the case or the pP15A, kanaR empty vector as the control, were transformed with the pTZ57R; the ability of the designed ZFN to disrupt the β-lactamase gene was evaluated with the subsequent disturbed ability of the bacteria to grow on ampicillin (amp) and ampicillin-kanamycin (amp-kana)-containing media. The effect of mild hypothermia on the ZFN gene targeting efficiency was also evaluated. Results: The growth of bacteria in the case group on the amp and amp-kana-containing media was significantly lower compared with the control group at 37°C (P < 0.001). Despite being more efficient in hypothermic conditions at 30°C (P < 0.001), there were no significant associations between the incubation temperature and the ZFN gene targeting efficiency. Conclusions: Our findings revealed that the ZFN technology could be employed to overcome ampicillin resistance by the targeted disruption of the ampicillin resistance gene, which leads to inactivation of β-lactam synthesis. Therefore, ZFN technology could be engaged to decrease the antibiotic resistance issue with the construction of a ZFN archive against different ARGs. To tackle the resistance issue at the environmental level, recombinant phages

Adjuvant chemoradiotherapy instead of revision radical resection after local excision for high-risk early rectal cancer.

Science.gov (United States)

Jeong, Jae-Uk; Nam, Taek-Keun; Kim, Hyeong-Rok; Shim, Hyun-Jeong; Kim, Yong-Hyub; Yoon, Mee Sun; Song, Ju-Young; Ahn, Sung-Ja; Chung, Woong-Ki

2016-09-05

After local excision of early rectal cancer, revision radical resection is recommended for patients with high-risk pathologic stage T1 (pT1) or pT2 cancer, but the revision procedure has high morbidity rates. We evaluated the efficacy of adjuvant concurrent chemoradiotherapy (CCRT) for reducing recurrence after local excision in these patients. Eighty-three patients with high-risk pT1 or pT2 rectal cancer underwent postoperative adjuvant CCRT after local excision. We defined high-risk features as pT1 having tumor size ≤3 cm, and/or resection margin (RM) ≤3 mm, and/or lymphovascular invasion (LVI), and/or non-full thickness excision such as endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD), or unknown records regarding those features, or pT2 cancer. Radiotherapy was administered with a median dose of 50.4 Gy in 1.8 Gy fraction size over 5-7 weeks. Concurrent 5-fluorouracil and leucovorin were administered for 4 days in the first and fifth weeks of radiotherapy. The median interval between local excision and radiotherapy was 34 (range, 11-104) days. Fifteen patients (18.1 %) had stage pT2 tumors, 22 (26.5 %) had RM of ≥3 mm, and 21 (25.3 %) had tumors of ≥3 cm in size. Thirteen patients (15.7 %) had LVI. Transanal excision was performed in 58 patients (69.9 %) and 25 patients (30.1 %) underwent EMR or ESD. The median follow-up was 61 months. The 5-year overall survival (OS), locoregional relapse-free survival (LRFS), and disease-free survival (DFS) rates for all patients were 94.9, 91.0, and 89.8 %, respectively. Multivariate analysis did not identify any significant factors for OS or LRFS, but the only significant factor affecting DFS was the pT stage (p = 0.027). In patients with high-risk pT1 rectal cancer, adjuvant CCRT after local excision could be an effective alternative treatment instead of revision radical resection. However, patients with pT2 stage showed inferior DFS compared to pT1.

Positive malignant margins in clinically diagnosed and excised be ...

African Journals Online (AJOL)

This study was aimed at utilizing retrospective descriptive data to evaluate the percentage of clini-cally benign breast lumps that turned out to be histologically malignant and the prevalence of posi-tive tumour margins among the malignant cases. A total of 2,917 registered cases of excised breast lump at the Department of ...

Regeneration of Surgically Excised Segments of Dog Esophagus using Biodegradable PLA Hollow Organ Grafts,

Science.gov (United States)

1980-06-01

7 AG 396 ARMY INST OF DENTAL RESEARCH WASHINGTON DC FIG 6/5 REGENERATION OF SURGICALLY EXCISED SEGMENTS OF DOG ESOPHAGUS US-ETC(W) U15 G’OE UN8 N F...the graft; infection; inadequate blood supply; difficulties in suture retention; leakage at the anastomatic sites; stenosis of the anasto- mosis...excised segment of the dog esophagus. On a conceptual L- J basis, the use of a biodegradable polymer to fabricate a successful J hollow organ graft holds

Siliconomas of the lower limb: Tumour-like excision and reconstruction

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Tommaso Agostini

2016-01-01

Full Text Available Background: Silicone oil injection can cause several complications including pain, cellulitis, abscesses, skin compromise, migration, embolism and multiorgan failure. Oil-infested tissue excision remains the solely treatment to prevent such complications. Objectives: The authors evaluate tumor-like excision of the oil-infested tissue as a treatment for patients experiencing silicone injections in the lower limbs, to both preserve aesthetic appearance and solve further risk of complication from silicone. Methods: Between January 2004 and January 2011 a total of 12 consecutive, nonrandomized female patients underwent surgical management of siliconoma of the lower limb. The mean age was 41-years, range from 22 to 61 years and all patients didn't referred comorbidities. Eight siliconomas were located on the leg and 4 were on the thigh. The mean area of siliconoma was 35 cm2, range from 25 to 60 cm2. Each patient was evaluated by ultrasonography and EchocolorDoppler of the soft tissue and in order to achieve a “staging” of siliconoma. Results: Healing was uneventful in all cases. Three patients (25% suffering hypertrophic scarring underwent further injection of corticosteroids to improve hypertrophic scars quality. Two patients (16.6% required a further session of structural fat grafting to improve thigh's profile. All patients were satisfied with the cosmetic results and indispositions requiring medical therapy disappeared. Conclusions: Tumor-like excision and immediate reconstruction appears to be a safe and consistent surgical option that preserves aesthetic appearance for patients victims of illegal oil silicone injection of the lower limbs.

A history of the DNA repair and mutagenesis field: The discovery of base excision repair.

Science.gov (United States)

Friedberg, Errol C

2016-01-01

This article reviews the early history of the discovery of an DNA repair pathway designated as base excision repair (BER), since in contrast to the enzyme-catalyzed removal of damaged bases from DNA as nucleotides [called nucleotide excision repair (NER)], BER involves the removal of damaged or inappropriate bases, such as the presence of uracil instead of thymine, from DNA as free bases. Copyright © 2015. Published by Elsevier B.V.

Developmentally-Regulated Excision of the SPβ Prophage Reconstitutes a Gene Required for Spore Envelope Maturation in Bacillus subtilis

Science.gov (United States)

Abe, Kimihiro; Kawano, Yuta; Iwamoto, Keito; Arai, Kenji; Maruyama, Yuki; Eichenberger, Patrick; Sato, Tsutomu

2014-01-01

Temperate phages infect bacteria by injecting their DNA into bacterial cells, where it becomes incorporated into the host genome as a prophage. In the genome of Bacillus subtilis 168, an active prophage, SPβ, is inserted into a polysaccharide synthesis gene, spsM. Here, we show that a rearrangement occurs during sporulation to reconstitute a functional composite spsM gene by precise excision of SPβ from the chromosome. SPβ excision requires a putative site-specific recombinase, SprA, and an accessory protein, SprB. A minimized SPβ, where all the SPβ genes were deleted, except sprA and sprB, retained the SPβ excision activity during sporulation, demonstrating that sprA and sprB are necessary and sufficient for the excision. While expression of sprA was observed during vegetative growth, sprB was induced during sporulation and upon mitomycin C treatment, which triggers the phage lytic cycle. We also demonstrated that overexpression of sprB (but not of sprA) resulted in SPβ prophage excision without triggering the lytic cycle. These results suggest that sprB is the factor that controls the timing of phage excision. Furthermore, we provide evidence that spsM is essential for the addition of polysaccharides to the spore envelope. The presence of polysaccharides on the spore surface renders the spore hydrophilic in water. This property may be beneficial in allowing spores to disperse in natural environments via water flow. A similar rearrangement occurs in Bacillus amyloliquefaciens FZB42, where a SPβ-like element is excised during sporulation to reconstitute a polysaccharide synthesis gene, suggesting that this type of gene rearrangement is common in spore-forming bacteria because it can be spread by phage infection. PMID:25299644

Teenage patients with ingrown toenails: Treatment with partial matrix excision or segmental phenolization

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Murat Korkmaz

2013-01-01

Full Text Available Background: Ingrown toenails (IT is a very common problem leading to significant associated morbidity. The articles related to phenolization for matrix removal in teenagers with IT are not enough in the foot surgery literature. Aims: To compare the postoperative recovery periods, complication rate, and tolerability of partial matrix excision and segmental phenolization in teenagers with IT. Materials and Methods: Thirty-nine patients (13-17 years with 48 IT were randomly divided into two groups and were treated with partial matrix excision (Group I and segmental phenolization (Group II. We assessed the recurrence rates, postoperative complications, duration of analgesic usage, and time to return to daily activities. Results: There was no significant difference between the demographic and clinical data of the two groups. Three patients in Group I and two patients in Group II experienced moderate pain postoperatively. These patients used analgesics for 3 days. The rates of postoperative complications and recurrences between the two groups showed no statistically significant difference ( P = 0.688. The time to return to normal daily activities was significantly shorter in Group II patients than in Group I patients ( P < 0.05. Conclusions: Partial matrix excision is a very safe model of therapy in the surgical treatment of teenagers with IT. It has low recurrence rate and minimal postoperative morbidity. We concluded that segmental phenolization is also as safe as partial matrix excision in the treatment of IT and patients return to their daily activities in less time with this treatment modality.

SUMO and ubiquitin-dependent XPC exchange drives nucleotide excision repair

DEFF Research Database (Denmark)

Van Cuijk, Loes; Van Belle, Gijsbert J.; Turkyilmaz, Yasemin

2015-01-01

XPC recognizes UV-induced DNA lesions and initiates their removal by nucleotide excision repair (NER). Damage recognition in NER is tightly controlled by ubiquitin and SUMO modifications. Recent studies have shown that the SUMO-targeted ubiquitin ligase RNF111 promotes K63-linked ubiquitylation o...

Early burn wound excision and skin grafting postburn trauma restores in vivo neutrophil delivery to inflammatory lesions

International Nuclear Information System (INIS)

Tchervenkov, J.I.; Epstein, M.D.; Silberstein, E.B.; Alexander, J.W.

1988-01-01

This study assessed the effect of early vs delayed postburn wound excision and skin grafting on the in vivo neutrophil delivery to a delayed-type hypersensitivity (DTH) reaction and a bacterial skin lesion (BSL). Male Lewis rats were presensitized to keyhole-limpet hemocyanin. Group 1 comprised sham controls. Groups 2 through 4 were given a 30% 3 degrees scald burn, but the burn wounds were excised, and skin was grafted on days 1, 3, and 7, respectively, after the burn. Group 5 comprised burn controls. Twelve days after burn trauma, all rats were injected at different intervals (during a 24-hour period) with a trio of intradermal injections of keyhole-limpet hemocyanin, Staphylococcus aureus 502A, and saline at different sites. In vivo neutrophil delivery to these dermal lesions was determined by injecting indium in 111 oxyquinoline-labeled neutrophils isolated from similarly treated groups of rats. Neutrophil delivery to DTH and BSL lesions was restored to normal by excision and skin grafting of the burn wound one day after burn trauma. Waiting three days after burn trauma to excise and skin graft the wound partially, but not completely, restored the in vivo neutrophil delivery to DTH and BSL lesions. Waiting one week to excise and skin graft a burn wound resulted in no improvement in neutrophil delivery to DTH and BSL dermal lesions. It was concluded that burn wound excision and skin grafting immediately after burn trauma restored in vivo neutrophil delivery to a BSL and DTH dermal lesion. This may, in part, explain the beneficial effect of early aggressive burn wound debridement in patients with burn injuries

A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease.

Science.gov (United States)

Sabzehei, Faezeh; Kouhpayeh, Shirin; Dastjerdeh, Mansoureh Shahbazi; Khanahmad, Hossein; Salehi, Rasoul; Naderi, Shamsi; Taghizadeh, Razieh; Rabiei, Parisa; Hejazi, Zahra; Shariati, Laleh

2017-01-01

Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods. At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F' that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry. The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) ( P < 0.0001). According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample.

Modeling base excision repair in Escherichia coli bacterial cells

International Nuclear Information System (INIS)

Belov, O.V.

2011-01-01

A model describing the key processes in Escherichia coli bacterial cells during base excision repair is developed. The mechanism is modeled of damaged base elimination involving formamidopyrimidine DNA glycosylase (the Fpg protein), which possesses several types of activities. The modeling of the transitions between DNA states is based on a stochastic approach to the chemical reaction description

A Rat Excised Larynx Model of Vocal Fold Scar

Science.gov (United States)

Welham, Nathan V.; Montequin, Douglas W.; Tateya, Ichiro; Tateya, Tomoko; Choi, Seong Hee; Bless, Diane M.

2009-01-01

Purpose: To develop and evaluate a rat excised larynx model for the measurement of acoustic, aerodynamic, and vocal fold vibratory changes resulting from vocal fold scar. Method: Twenty-four 4-month-old male Sprague-Dawley rats were assigned to 1 of 4 experimental groups: chronic vocal fold scar, chronic vocal fold scar treated with 100-ng basic…

Excision Les jeunes changent l'Afrique par les TIC

International Development Research Centre (IDRC) Digital Library (Canada)

Comité Inter-Africain sur les Pratiques Traditionnelles ayant effet sur la santé des ... Planche 7 - Perception genrée de la citoyenneté : les dire des femmes. 90 ...... dans le monde avec les migrations internationales, l'excision ne semble pas être ...... En utilisant les TIC pour exprimer leurs attentes et leurs besoins, les jeunes, ...

Combined laparoscopic abdomino-endoscopic perineal total mesorectal excision for anorectal malignant melanoma: A case report

Directory of Open Access Journals (Sweden)

Ryo Ohta

Full Text Available Introduction: This report presents a case of anorectal malignant melanoma treated with combined laparoscopic abdomino-endoscopic perineal total mesorectal excision. Presentation of case: An 82-year-old female presented with hematochezia. Colonoscopy revealed a 5-cm tumor in the anorectal junction, and biopsy specimen showed malignant melanoma. Modified ransanal total mesorectal excision was performed to get the sufficient surgical resection margins. After lymph node dissection in usual manner, mobilizing the rectum to the level of levator ani muscle. Then a skin incision was made around the anus and the transperineal access platform was placed. The fat tissue of the ischioanal fossa was divided until the levator ani muscle was exposed. The oral side of the colon was transected and specimen was extracted through the perineal incision site. Then stoma was placed laparoscopically. Discussion: This procedure provides not only better exposure of the extralevator surgical field, but also efficient resection margins compared with the conventional andominoperineal resection. Conclusion: To the best of our knowledge, this is the first report of combined laparoscopic abdomino-endoscopic perineal total mesorectal excision for anorectal malignant melanoma. Our experience showed safety and feasible option for anorectal malignant diseases. Keywords: Anorectal malignant melanoma, Transanal total mesorectal excision, Laparoscopic abdominoperineal resection, Case report

Harman inhibits the removal of pyrimidine dimers from the DNA of human cells

International Nuclear Information System (INIS)

Castellani, A.; Setlow, R.B.

1981-01-01

Normal human fibroblasts were UV-irradiated and incubated for 6 hr with harman. The losses of sites, in the extracted DNA, sensitive to a UV specific endonuclease were determined as precision measures of the excision of UV-induced pyrimidine dimers. Harman inhibited excision, rising from approx. 30% inhibition at 200 μM to 75% inhibition at 500 μM

Probing force-induced unfolding intermediates of a single staphylococcal nuclease molecule and the effect of ligand binding

International Nuclear Information System (INIS)

Ishii, Takaaki; Murayama, Yoshihiro; Katano, Atsuto; Maki, Kosuke; Kuwajima, Kunihiro; Sano, Masaki

2008-01-01

Single-molecule manipulation techniques have given experimental access to unfolding intermediates of proteins that are inaccessible in conventional experiments. A detailed characterization of the intermediates is a challenging problem that provides new possibilities for directly probing the energy landscape of proteins. We investigated single-molecule mechanical unfolding of a small globular protein, staphylococcal nuclease (SNase), using atomic force microscopy. The unfolding trajectories of the protein displayed sub-molecular and stochastic behavior with typical lengths corresponding to the size of the unfolded substructures. Our results support the view that the single protein unfolds along multiple pathways as suggested in recent theoretical studies. Moreover, we found the drastic change, caused by the ligand and inhibitor bindings, in the mechanical unfolding dynamics

Distribution of nuclease attack sites and complexity of DNA in the products of post-irradiation degradiation of rat thymus chromatin

International Nuclear Information System (INIS)

Zvonareva, N.B.; Zhivotovsky, B.D.; Hanson, K.P.

1983-01-01

The distribution of nuclease attack sites in chromatin has been studied on the basis of the quantitative relationship of the single- and double-stranded fragments of various lengths in the products of post-irradiation degradation of chromatin (PDN). It has been shown that in irradiated thymocytes internucleosome degradation of chromatin occurs and the products of the enzymic digestion of chromatin derive from randomly distributed genome areas accumulate. Analysis of the reassociation curves has not shown any differences in the complexity of the PDN fractions and total DNA. (author)

76 FR 46677 - Indoor Tanning Services; Cosmetic Services Excise Taxes

Science.gov (United States)

2011-08-03

... DEPARTMENT OF THE TREASURY Internal Revenue Service 26 CFR Parts 40 and 49 [REG-112841-10] RIN 1545-BJ40 Indoor Tanning Services; Cosmetic Services Excise Taxes AGENCY: Internal Revenue Service (IRS), Treasury. ACTION: Notice of public hearing on proposed rulemaking. SUMMARY: This document provides notice...

Metabolic modulation of mammalian DNA excision repair

Energy Technology Data Exchange (ETDEWEB)

Schrader, T.J.

1988-01-01

First, ultraviolet light (UVL)- and dimethylsulfate (DMS)-induced excision repair was examined in quiescent and lectin-stimulated bovine lymphocytes. Upon mitogenic stimulation, UVL-induced repair increased by a factor of 2 to 3, and reached this maximum 2 days before the onset of DNA replication. However, DMS-induced repair increased sevenfold in parallel with DNA replication. Repair patch sizes were smaller for DMS-induced damage reflecting patches of 7 nucleotides in quiescent lymphocytes compared to 20 nucleotides induced by UVL. The patch size increased during lymphocyte stimulation until one day prior to the peak of DNA replication when patch sizes of 45 and 35 nucleotides were produced in response to UVL- and DMS-induced damage, respectively. At the peak of DNA replication, the patch sizes were equal for both damaging agents at 34 nucleotides. In the second study, a small amount of repair replication was observed in undamaged quiescent and concanavalin A-stimulated bovine lymphocytes as well as in human T98G glioblastoma cells. Repair incorporation doubled in the presence of hydroxyurea. Thirdly, the enhanced repair replication induced by the poly (ADP-ribose) polymerase inhibitor, 3-aminobenzamide, (3-AB), could not be correlated either with an increased rate of repair in the presence of 3-AB or with the use of hydroxyurea in the repair protocol. Finally, treatment of unstimulated lymphocytes with hyperthermia was accompanied by decreased repair replication while the repair patches remained constant at 20 nucleotides.

Stuck in Neutral: Stalled Progress in Statewide Comprehensive Smoke-Free Laws and Cigarette Excise Taxes, United States, 2000–2014

Science.gov (United States)

King, Brian A.; Babb, Stephen D.

2016-01-01

Introduction Increasing tobacco excise taxes and implementing comprehensive smoke-free laws are two of the most effective population-level strategies to reduce tobacco use, prevent tobacco use initiation, and protect nonsmokers from secondhand smoke. We examined state laws related to smoke-free buildings and to cigarette excise taxes from 2000 through 2014 to see how implementation of these laws from 2000 through 2009 differs from implementation in more recent years (2010–2014). Methods We used legislative data from LexisNexis, an online legal research database, to examine changes in statewide smoke-free laws and cigarette excise taxes in effect from January 1, 2000, through December 31, 2014. A comprehensive smoke-free law was defined as a statewide law prohibiting smoking in all indoor areas of private work sites, restaurants, and bars. Results From 2000 through 2009, 21 states and the District of Columbia implemented comprehensive smoke-free laws prohibiting smoking in work sites, restaurants, and bars. In 2010, 4 states implemented comprehensive smoke-free laws. The last state to implement a comprehensive smoke-free law was North Dakota in 2012, bringing the total number to 26 states and the District of Columbia. From 2000 through 2009, 46 states and the District of Columbia implemented laws increasing their cigarette excise tax, which increased the national average state excise tax rate by $0.92. However, from 2010 through 2014, only 14 states and the District of Columbia increased their excise tax, which increased the national average state excise tax rate by $0.20. Conclusion The recent stall in progress in enacting and implementing statewide comprehensive smoke-free laws and increasing cigarette excise taxes may undermine tobacco prevention and control efforts in the United States, undercutting efforts to reduce tobacco use, exposure to secondhand smoke, health disparities, and tobacco-related illness and death. PMID:27309417

Role of DNA lesions and repair in the transformation of human cells

International Nuclear Information System (INIS)

Maher, V.M.; McCormick, J.J.

1987-01-01

Results of studies on the transformation of diploid human fibroblasts in culture into tumor-forming cells by exposure to chemical carcinogens or radiation indicate that such transformation is multi-stepped process that at least one step, acquisition of anchorage independence, occurs as a mutagenic event. Studies comparing normal-repairing human cells with DNA repair-deficient cells, such as those derived from cancer-prone xeroderma pigmentosum patients, indicate that excision repair in human fibroblasts is essentially an error-free process that the ability to excise potentially cytotoxic, mutagenic, or transforming lesions induced DNA by carcinogens determines their ultimate biological consequences. Cells deficient in excision repair are abnormally sensitive to these agents. Studies with cells treated at various times in the cell cycle show that there is a certain limited amount of time available for DNA repair between the initial exposure and the onset of the cellular event responsible for mutation induction and transformation to anchorage independence. The data suggest that DNA replication on a template containing unexcised lesions (photoproducts, adducts) is the critical event

Clinicopathologic Significance of Excision Repair Cross-Complementation 1 Expression in Patients Treated With Breast-Conserving Surgery and Radiation Therapy

International Nuclear Information System (INIS)

Goyal, Sharad; Parikh, Rahul R.; Green, Camille; Schiff, Devora B.S.; Moran, Meena S.; Yang Qifeng; Haffty, Bruce G.

2010-01-01

Purpose: The excision repair cross-complementation 1 (ERCC1) enzyme plays a rate-limiting role in the nucleotide excision repair pathway and is associated with resistance to platinum-based chemotherapy in cancers of the head and neck and the lung. The purpose of this study was to evaluate the clinicopathologic and prognostic significance of ERCC1 expression in a cohort of early-stage breast cancer patients treated with breast conservation therapy. Methods and Materials: Paraffin specimens from 504 women with early-stage breast cancer treated with breast conservation therapy were constructed into tissue microarrays. The array was stained for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) and ERCC1. This was then correlated with clinicopathologic factors and outcomes data. Results: ERCC-1 expression was evaluable in 366 cases (72%). In this group, 32% and 38% of patients received adjuvant chemotherapy and hormonal therapy, respectively. Increased ERCC-1 expression was found to be correlated with ER positivity (p 50 (p 50. To our knowledge, this is the first study investigating ERCC1 expression in patients receiving adjuvant radiation therapy for breast cancer.

Genetic instability associated with loop or stem–loop structures within transcription units can be independent of nucleotide excision repair

Science.gov (United States)

Burns, John A; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Scicchitano, David A

2018-01-01

Abstract Simple sequence repeats (SSRs) are found throughout the genome, and under some conditions can change in length over time. Germline and somatic expansions of trinucleotide repeats are associated with a series of severely disabling illnesses, including Huntington's disease. The underlying mechanisms that effect SSR expansions and contractions have been experimentally elusive, but models suggesting a role for DNA repair have been proposed, in particular the involvement of transcription-coupled nucleotide excision repair (TCNER) that removes transcription-blocking DNA damage from the transcribed strand of actively expressed genes. If the formation of secondary DNA structures that are associated with SSRs were to block RNA polymerase progression, TCNER could be activated, resulting in the removal of the aberrant structure and a concomitant change in the region's length. To test this, TCNER activity in primary human fibroblasts was assessed on defined DNA substrates containing extrahelical DNA loops that lack discernible internal base pairs or DNA stem–loops that contain base pairs within the stem. The results show that both structures impede transcription elongation, but there is no corresponding evidence that nucleotide excision repair (NER) or TCNER operates to remove them. PMID:29474673

26 CFR 54.4975-14 - Election to pay an excise tax for certain pre-1975 prohibited transactions.

Science.gov (United States)

2010-04-01

...-14 Election to pay an excise tax for certain pre-1975 prohibited transactions. (a) In general... the district director to extend the time within which the election is permitted. (d) Computation of... 26 Internal Revenue 17 2010-04-01 2010-04-01 false Election to pay an excise tax for certain pre...

Efficacy of systemic adjuvant therapies administered to dogs after excision of oral malignant melanomas: 151 cases (2001-2012).

Science.gov (United States)

Boston, Sarah E; Lu, Xiaomin; Culp, William T N; Montinaro, Vincenzo; Romanelli, Giorgio; Dudley, Robert M; Liptak, Julius M; Mestrinho, Lisa A; Buracco, Paolo

2014-08-15

To determine prognostic factors for and compare outcome among dogs with oral malignant melanoma following excision with or without various systemic adjuvant therapies. Retrospective case series. 151 dogs with naturally occurring oral malignant melanomas treated by excision with or without adjuvant therapies from 2001 to 2012. Case accrual was solicited from Veterinary Society of Surgical Oncology members via an email list service. Information collected from case records included signalment, tumor staging, tumor characteristics, type of surgical excision, histologic diagnosis, adjuvant therapy, and survival time. The overall median survival time was 346 days. Results of multivariate analysis indicated that tumor size, patient age, and intralesional excision (vs marginal, wide, or radical excision) were considered poor prognostic indicators. All other demographic and clinical variables were not significantly associated with survival time after adjusting for the aforementioned 3 variables. A clear survival benefit was not evident with any systemic adjuvant therapy, including vaccination against melanoma or chemotherapy; however, the number of dogs in each treatment group was small. Ninety-eight dogs received no postoperative adjuvant therapy, and there was no difference in survival time between dogs that did (335 days) and did not (352 days) receive systemic adjuvant therapy. For dogs with oral malignant melanoma, increasing tumor size and age were negative prognostic factors. Complete excision of all macroscopic tumor burden improved survival time. Long-term survival was possible following surgery alone. Although systemic adjuvant therapy was not found to improve survival time, this could have been due to type II error.

Unscheduled DNA synthesis in human skin after in vitro ultraviolet-excimer laser ablation

International Nuclear Information System (INIS)

Green, H.A.; Margolis, R.; Boll, J.; Kochevar, I.E.; Parrish, J.A.; Oseroff, A.R.

1987-01-01

DNA damage repaired by the excision repair system and measured as unscheduled DNA synthesis (UDS) was assessed in freshly excised human skin after 193 and 248 nm ultraviolet (UV)-excimer laser ablative incisions. Laser irradiation at 248 nm induced DNA damage throughout a zone of cells surrounding the ablated and heat-damaged area. In contrast, with 193 nm irradiation UDS was not detected in cells adjacent to the ablated area, even though DNA strongly absorbs this wavelength. Our results suggest that the lack of UDS after 193 nm irradiation is due to: ''shielding'' of DNA by the cellular interstitium, membrane, and cytoplasm, DNA damage that is not repaired by excision repair, or thermal effects that either temporarily or permanently inhibit the excision repair processes

Patient satisfaction and efficacy of vacuum-assisted excision biopsy of fibroadenomas

Energy Technology Data Exchange (ETDEWEB)

Thurley, P. [Nottingham Breast Institute, Nottingham University Hospitals NHS Trust, Nottingham (United Kingdom)], E-mail: pthurley@doctors.org.uk; Evans, A.; Hamilton, L.; James, J.; Wilson, R. [Nottingham Breast Institute, Nottingham University Hospitals NHS Trust, Nottingham (United Kingdom)

2009-04-15

Aim: To establish the efficacy, complications, and patient satisfaction for vacuum-assisted biopsy (VAB) excision of fibroadenomas. Materials and methods: All patients referred for VAB of benign lesions from 11 December 2002 to 30 November 2006 were identified prospectively, and the following data were recorded: age, histology, lesion size, and completeness of excision. A questionnaire was sent at least a year after the procedure to assess pain, complications, residual palpable lesions, and cosmetic result. Results: One hundred and thirty-four patients were referred for VAB, 81 had fibroadenomas. Fifty-nine percent replied to the questionnaire. Fifty-four percent of patients reported no pain during the procedure, 8% rated their pain at {>=}3/10. Thirty-two percent had no pain the week after the procedure, 55% had pain {>=}3/10. Seventy-nine percent had no palpable mass at the site of the original lesion. Ninety-four percent would recommend the procedure to others and would prefer further VAB to surgery. Bruising was common, one patient required aspiration of a haematoma. Six percent developed infections, all resolved with antibiotic therapy. Eighty-five percent of patients were completely satisfied with the cosmetic result. Interval ultrasound was performed in 36 patients. A palpable mass was present in 11%, a non-palpable mass in 19%, and no mass in 70%. Conclusion: VAB excision is well-tolerated, safe, and popular with a high initial success rate for fibroadenomas. Bruising and pain are common the week after the procedure.

Patient satisfaction and efficacy of vacuum-assisted excision biopsy of fibroadenomas

International Nuclear Information System (INIS)

Thurley, P.; Evans, A.; Hamilton, L.; James, J.; Wilson, R.

2009-01-01

Aim: To establish the efficacy, complications, and patient satisfaction for vacuum-assisted biopsy (VAB) excision of fibroadenomas. Materials and methods: All patients referred for VAB of benign lesions from 11 December 2002 to 30 November 2006 were identified prospectively, and the following data were recorded: age, histology, lesion size, and completeness of excision. A questionnaire was sent at least a year after the procedure to assess pain, complications, residual palpable lesions, and cosmetic result. Results: One hundred and thirty-four patients were referred for VAB, 81 had fibroadenomas. Fifty-nine percent replied to the questionnaire. Fifty-four percent of patients reported no pain during the procedure, 8% rated their pain at ≥3/10. Thirty-two percent had no pain the week after the procedure, 55% had pain ≥3/10. Seventy-nine percent had no palpable mass at the site of the original lesion. Ninety-four percent would recommend the procedure to others and would prefer further VAB to surgery. Bruising was common, one patient required aspiration of a haematoma. Six percent developed infections, all resolved with antibiotic therapy. Eighty-five percent of patients were completely satisfied with the cosmetic result. Interval ultrasound was performed in 36 patients. A palpable mass was present in 11%, a non-palpable mass in 19%, and no mass in 70%. Conclusion: VAB excision is well-tolerated, safe, and popular with a high initial success rate for fibroadenomas. Bruising and pain are common the week after the procedure

of lung metastases carcinoma of the cervix Surgical excision from ...

African Journals Online (AJOL)

of lung metastases carcinoma of the cervix. Surgical excision from squamous. A report of 2 cases. N. G. DE MOOR, A. V. BERRY, M. M. NISSENBAUM. These2casereportsservetoemphasizetwoimpor- tant points concerning carcinoma of the cervix: (i) blood-borne metastases are now frequently encountered in this disease; ...

A Method To Determine Adhesion Of Suppository Mass On Excised ...

African Journals Online (AJOL)

A method to determine adhesion of suppository mass to intestinal tissue was developed using excised pig intestine. The method which employs the principe of drainage unto and subsequent detachment from the mucosa, of an adherent suppository mass is simple, inexpensive and accurate. Fully optimised, it can be used ...

Evaluation of type II thyroplasty on phonatory physiology in an excised canine larynx model

Science.gov (United States)

Devine, Erin E.; Hoffman, Matthew R.; McCulloch, Timothy M.; Jiang, Jack J.

2016-01-01

Objective Type II thyroplasty is an alternative treatment for spasmodic dysphonia, addressing hyperadduction by incising and lateralizing the thyroid cartilage. We quantified the effect of lateralization width on phonatory physiology using excised canine larynges. Methods Normal closure, hyperadduction, and type II thyroplasty (lateralized up to 5mm at 1mm increments with hyperadducted arytenoids) were simulated in excised larynges (N=7). Aerodynamic, acoustic, and videokymographic data were recorded at three subglottal pressures relative to phonation threshold pressure (PTP). One-way repeated measures ANOVA assessed effect of condition on aerodynamic parameters. Random intercepts linear mixed effects models assessed effects of condition and subglottal pressure on acoustic and videokymographic parameters. Results PTP differed across conditions (p<0.001). Condition affected percent shimmer (p<0.005) but not percent jitter. Both pressure (p<0.03) and condition (p<0.001) affected fundamental frequency. Pressure affected vibratory amplitude (p<0.05) and intra-fold phase difference (p<0.05). Condition affected phase difference between the vocal folds (p<0.001). Conclusions Hyperadduction increased PTP and worsened perturbation compared to normal, with near normal physiology restored with 1mm lateralization. Further lateralization deteriorated voice quality and increased PTP. Acoustic and videokymographic results indicate that normal physiologic relationships between subglottal pressure and vibration are preserved at optimal lateralization width, but then degrade with further lateralization. The 1mm optimal width observed here is due to the small canine larynx size. Future human trials would likely demonstrate a greater optimal width, with patient-specific value potentially determined based on larynx size and symptom severity. PMID:27223665

Transanal endoscopic microsurgery versus conventional transanal excision for patients with early rectal cancer.

Science.gov (United States)

Christoforidis, Dimitrios; Cho, Hyeon-Min; Dixon, Matthew R; Mellgren, Anders F; Madoff, Robert D; Finne, Charles O

2009-05-01

To compare transanal endoscopic microsurgery (TEMS) with conventional transanal excision (TAE) in terms of the quality of resection, local recurrence, and survival rates in patients with stage I rectal cancer. Although TEMS is often considered a superior surgical technique to TAE, it is poorly suited for excising tumors in the lower third of the rectum. Such tumors may confer a worse prognosis. We retrospectively reviewed information on all patients with stage pT1 and pT2 rectal adenocarcinoma who underwent local excision from 1997 through mid-2006. We excluded patients with node-positive, metastatic, recurrent, previously irradiated, or snare-excised tumors. Our study included 42 TEMS and 129 TAE patients. We found no significant differences in patient characteristics, adjuvant therapy, tumor stage, or adverse histopathologic features. In the TAE group, 52 (40%) of tumors were TEMS group, only 1 (2%) (P = 0.0001). Surgical margins were less often positive in the TEMS group (2%) than in the TAE group (16%) (P = 0.017). For patients with tumors > or =5 cm from the AV, the estimated 5-year disease-free survival (DFS) rate was similar between the TEMS group (84.1%) and the TAE group (76.1%) (P = 0.651). But within the TAE group, the estimated 5-year DFS rate was better for patients with tumors > or =5 cm from the AV (76.1%) vs. TEMS or TAE) itself--were independent predictors of local recurrence and DFS. The quality of resection is better with TEMS than with TAE. However, the apparently better oncologic outcomes with TEMS can be partly explained by case selection of lower-risk tumors of the upper rectum.

Principles of Periocular Reconstruction following Excision of Cutaneous Malignancy

International Nuclear Information System (INIS)

Hayano, S. M.; Whipple, K. M.; Korn, B. S.; Kikkawa, D. O.

2012-01-01

Reconstruction of periocular defects following excision of cutaneous malignancy can present difficulties for oculofacial and reconstructive surgeons. The intricate anatomy of the eyelids and face requires precise restoration in order to avoid postoperative functional anesthetic concerns. Various reconstructive procedures based on common principles, location and size of the defect, can be applied to achieve restoration with the best possible functional and aesthetic outcomes.

Schwannoma in the porta hepatis - laparoscopic excision under laparoscopic ultrasound guidance.

Science.gov (United States)

Sebastian, Maciej; Sroczyński, Maciej; Donizy, Piotr; Rudnicki, Jerzy

2017-09-01

Schwannomas are usually benign tumors attached to peripheral nerves and are rarely found in the gastrointestinal tract. Schwannomas in the porta hepatis are extremely rare, with only 15 cases described in the literature to date. A 22-year-old female patient presented with colicky upper abdominal pain lasting 3 months. Magnetic resonance imaging of the abdominal cavity revealed a tumor in the porta hepatis. The patient was qualified for laparoscopy. The tumor was totally excised laparoscopically under guidance of laparoscopic ultrasound without intra- or postoperative complications. Postoperative histopathological examination confirmed the porta hepatic schwannoma. The patient recovered uneventfully with very good cosmetic results. In the follow-up period of 5 months we have not observed any abdominal or general health problems. The present case is the first report in the world of laparoscopic ultrasound guided laparoscopic excision of a porta hepatic schwannoma.

Repair of human DNA in molecules that replicate or remain unreplicated following ultraviolet irradiation

International Nuclear Information System (INIS)

Waters, R.

1980-01-01

The extent of DNA replication, the incidence of uv induced pyrimidine dimers and the repair replication observed after their excision was monitored in human fibroblasts uv irradiated with single or split uv doses. The excision repair processes were measured in molecules that remained unreplicated or in those that replicated after the latter uv irradiation. Less DNA replication was observed after a split as opposed to single uv irradiation. Furthermore, a split dose did not modify the excision parameters measured after a single irradiation, regardless of whether the DNA had replicated or not

Descriptive Study of Patients Receiving Excision and Radiotherapy for Keloids

International Nuclear Information System (INIS)

Speranza, Giovanna; Sultanem, Khalil M.D.; Muanza, Thierry

2008-01-01

Purpose: To review and describe our institution's outcomes in patients treated with external beam radiotherapy after keloid excision. Methods and Materials: This was a retrospective study. Patients who received radiotherapy between July 1994 and January 2004 after keloid excision were identified. A questionnaire was mailed regarding sociodemographic factors, early and late radiation toxicities, the need for additional therapy, and satisfaction level. All patients had received a total of 15 Gy in three daily 5-Gy fractions. Treatment started within 24 h after surgery and was delivered on a Siemens orthovoltage machine. The data were analyzed using the STATA statistical package. Results: A total of 234 patients were approached. The response rate was 41%, and 75% were female. The mean age was 36.5 years (range, 16-69 years). The patients were mainly of European (53.1%) or African (19.8%) descent. For early toxicity outcomes, 54.2% reported skin redness and 24% reported skin peeling. For late toxicity outcomes, 27% reported telangiectasia and 62% reported permanent skin color changes. No association was found with gender, skin color, or age for the late toxicity outcomes. Of the patients responding, 14.6% required adjuvant treatment. On a visual scale of 1-10 for the satisfaction level, 60% reported a satisfaction level of ≥8. Telangiectasia was the most significant predictor of a low satisfaction level (≤3, p < 0.005). Conclusion: The results of our study have shown that orthovoltage-based radiotherapy after surgical excision for keloids is a good method for the prevention of relapse. It is well tolerated, causes little toxicity, and leads to a high patient satisfaction level