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Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway

KAUST Repository

Zaher, Manal S.; Rashid, Fahad; Song, Bo; Joudeh, Luay I; Sobhy, Mohamed Abdelmaboud; Tehseen, Muhammad; Hingorani, Manju M; Hamdan, Samir

2018-01-01

RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5'-single-stranded flap is cleaved by structure-specific 5'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.
Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway

KAUST Repository

Zaher, Manal S.

2018-01-27

RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5\\'-single-stranded flap is cleaved by structure-specific 5\\'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.
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.
Generation of knockout rabbits using transcription activator-like effector nucleases.

Science.gov (United States)

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

2014-01-01

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

Directory of Open Access Journals (Sweden)

Yu Wang

2014-01-01

Full Text Available Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.
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 ...
Rate-determining Step of Flap Endonuclease 1 (FEN1) Reflects a Kinetic Bias against Long Flaps and Trinucleotide Repeat Sequences.

Science.gov (United States)

Tarantino, Mary E; Bilotti, Katharina; Huang, Ji; Delaney, Sarah

2015-08-21

Flap endonuclease 1 (FEN1) is a structure-specific nuclease responsible for removing 5'-flaps formed during Okazaki fragment maturation and long patch base excision repair. In this work, we use rapid quench flow techniques to examine the rates of 5'-flap removal on DNA substrates of varying length and sequence. Of particular interest are flaps containing trinucleotide repeats (TNR), which have been proposed to affect FEN1 activity and cause genetic instability. We report that FEN1 processes substrates containing flaps of 30 nucleotides or fewer at comparable single-turnover rates. However, for flaps longer than 30 nucleotides, FEN1 kinetically discriminates substrates based on flap length and flap sequence. In particular, FEN1 removes flaps containing TNR sequences at a rate slower than mixed sequence flaps of the same length. Furthermore, multiple-turnover kinetic analysis reveals that the rate-determining step of FEN1 switches as a function of flap length from product release to chemistry (or a step prior to chemistry). These results provide a kinetic perspective on the role of FEN1 in DNA replication and repair and contribute to our understanding of FEN1 in mediating genetic instability of TNR sequences. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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
Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1.

Science.gov (United States)

Gloor, Jason W; Balakrishnan, Lata; Campbell, Judith L; Bambara, Robert A

2012-08-01

In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼ 30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.
Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair.

Science.gov (United States)

Warbrick, E; Lane, D P; Glover, D M; Cox, L S

1997-05-15

Following genomic damage, the cessation of DNA replication is co-ordinated with onset of DNA repair; this co-ordination is essential to avoid mutation and genomic instability. To investigate these phenomena, we have analysed proteins that interact with PCNA, which is required for both DNA replication and repair. One such protein is p21Cip1, which inhibits DNA replication through its interaction with PCNA, while allowing repair to continue. We have identified an interaction between PCNA and the structure specific nuclease, Fen1, which is involved in DNA replication. Deletion analysis suggests that p21Cip1 and Fen1 bind to the same region of PCNA. Within Fen1 and its homologues a small region (10 amino acids) is sufficient for PCNA binding, which contains an 8 amino acid conserved PCNA-binding motif. This motif shares critical residues with the PCNA-binding region of p21Cip1. A PCNA binding peptide from p21Cip1 competes with Fen1 peptides for binding to PCNA, disrupts the Fen1-PCNA complex in replicating cell extracts, and concomitantly inhibits DNA synthesis. Competition between homologous regions of Fen1 and p21Cip1 for binding to the same site on PCNA may provide a mechanism to co-ordinate the functions of PCNA in DNA replication and repair.
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.
Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs.

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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.
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...
Distinct Mechanisms of Nuclease-Directed DNA-Structure-Induced Genetic Instability in Cancer Genomes.

Science.gov (United States)

Zhao, Junhua; Wang, Guliang; Del Mundo, Imee M; McKinney, Jennifer A; Lu, Xiuli; Bacolla, Albino; Boulware, Stephen B; Zhang, Changsheng; Zhang, Haihua; Ren, Pengyu; Freudenreich, Catherine H; Vasquez, Karen M

2018-01-30

Sequences with the capacity to adopt alternative DNA structures have been implicated in cancer etiology; however, the mechanisms are unclear. For example, H-DNA-forming sequences within oncogenes have been shown to stimulate genetic instability in mammals. Here, we report that H-DNA-forming sequences are enriched at translocation breakpoints in human cancer genomes, further implicating them in cancer etiology. H-DNA-induced mutations were suppressed in human cells deficient in the nucleotide excision repair nucleases, ERCC1-XPF and XPG, but were stimulated in cells deficient in FEN1, a replication-related endonuclease. Further, we found that these nucleases cleaved H-DNA conformations, and the interactions of modeled H-DNA with ERCC1-XPF, XPG, and FEN1 proteins were explored at the sub-molecular level. The results suggest mechanisms of genetic instability triggered by H-DNA through distinct structure-specific, cleavage-based replication-independent and replication-dependent pathways, providing critical evidence for a role of the DNA structure itself in the etiology of cancer and other human diseases. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
Small molecule inhibitors uncover synthetic genetic interactions of human flap endonuclease 1 (FEN1 with DNA damage response genes.

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Thomas A Ward

Full Text Available Flap endonuclease 1 (FEN1 is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption.
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
Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

KAUST Repository

Rashid, Fahad

2017-02-23

Human flap endonuclease 1 (FEN1) and related structure-specific 5\\'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5\\'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually \\'locks\\' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.
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.
Interaction between APC and Fen1 during breast carcinogenesis.

Science.gov (United States)

Narayan, Satya; Jaiswal, Aruna S; Law, Brian K; Kamal, Mohammad A; Sharma, Arun K; Hromas, Robert A

2016-05-01

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER - adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) - promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and
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.

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.
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)
Genome Editing in Rats Using TALE Nucleases.

Science.gov (United States)

Tesson, Laurent; Remy, Séverine; Ménoret, Séverine; Usal, Claire; Thinard, Reynald; Savignard, Chloé; De Cian, Anne; Giovannangeli, Carine; Concordet, Jean-Paul; Anegon, Ignacio

2016-01-01

The rat is an important animal model to understand gene function and model human diseases. Since recent years, the development of gene-specific nucleases has become important for generating new rat models of human diseases, to analyze the role of genes and to generate human antibodies. Transcription activator-like (TALE) nucleases efficiently create gene-specific knockout rats and lead to the possibility of gene targeting by homology-directed recombination (HDR) and generating knock-in rats. We describe a detailed protocol for generating knockout and knock-in rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.
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
Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand*

Science.gov (United States)

Teasley, Daniel C.; Parajuli, Shankar; Nguyen, Mai; Moore, Hayley R.; Alspach, Elise; Lock, Ying Jie; Honaker, Yuchi; Saharia, Abhishek; Piwnica-Worms, Helen; Stewart, Sheila A.

2015-01-01

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer. PMID:25922071
Human FEN1 Expression and Solubility Patterson in DNA Replication and Repair

National Research Council Canada - National Science Library

Carrier, Richard

1999-01-01

Flap endo-/exonuclease (FEN1) is a highly conserved protein shown to be one of 10 essential human proteins required for the production of form I DNA following DNA replication from the simian virus 40 (SV40...
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.
The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis.

Science.gov (United States)

Burkhart, Brett W; Cubonova, Lubomira; Heider, Margaret R; Kelman, Zvi; Reeve, John N; Santangelo, Thomas J

2017-07-01

Many aspects of and factors required for DNA replication are conserved across all three domains of life, but there are some significant differences surrounding lagging-strand synthesis. In Archaea , a 5'-to-3' exonuclease, related to both bacterial RecJ and eukaryotic Cdc45, that associates with the replisome specifically through interactions with GINS was identified and designated GAN (for G INS- a ssociated n uclease). Despite the presence of a well-characterized flap endonuclease (Fen1), it was hypothesized that GAN might participate in primer removal during Okazaki fragment maturation, and as a Cdc45 homologue, GAN might also be a structural component of an archaeal CMG (Cdc45, MCM, and GINS) replication complex. We demonstrate here that, individually, either Fen1 or GAN can be deleted, with no discernible effects on viability and growth. However, deletion of both Fen1 and GAN was not possible, consistent with both enzymes catalyzing the same step in primer removal from Okazaki fragments in vivo RNase HII has also been proposed to participate in primer processing during Okazaki fragment maturation. Strains with both Fen1 and RNase HII deleted grew well. GAN activity is therefore sufficient for viability in the absence of both RNase HII and Fen1, but it was not possible to construct a strain with both RNase HII and GAN deleted. Fen1 alone is therefore insufficient for viability in the absence of both RNase HII and GAN. The ability to delete GAN demonstrates that GAN is not required for the activation or stability of the archaeal MCM replicative helicase. IMPORTANCE The mechanisms used to remove primer sequences from Okazaki fragments during lagging-strand DNA replication differ in the biological domains. Bacteria use the exonuclease activity of DNA polymerase I, whereas eukaryotes and archaea encode a flap endonuclease (Fen1) that cleaves displaced primer sequences. RNase HII and the GINS-associated exonuclease GAN have also been hypothesized to assist in primer
Seed dispersal in fens

Science.gov (United States)

Middleton, B.; Van Diggelen, R.; Jensen, K.

2006-01-01

Question: How does seed dispersal reduce fen isolation and contribute to biodiversity? Location: European and North American fens. Methods: This paper reviews the literature on seed dispersal to fens. Results: Landscape fragmentation may reduce dispersal opportunities thereby isolating fens and reducing genetic exchange. Species in fragmented wetlands may have lower reproductive success, which can lead to biodiversity loss. While fens may have always been relatively isolated from each other, they have become increasingly fragmented in modern times within agricultural and urban landscapes in both Europe and North America. Dispersal by water, animals and wind has been hampered by changes related to development in landscapes surrounding fens. Because the seeds of certain species are long-lived in the seed bank, frequent episodes of dispersal are not always necessary to maintain the biodiversity of fens. However, of particular concern to restoration is that some dominant species, such as the tussock sedge Carex stricta, may not disperse readily between fens. Conclusions: Knowledge of seed dispersal can be used to maintain and restore the biodiversity of fens in fragmented landscapes. Given that development has fragmented landscapes and that this situation is not likely to change, the dispersal of seeds might be enhanced by moving hay or cattle from fens to damaged sites, or by reestablishing lost hydrological connections. ?? IAVS; Opulus Press.
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.
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.
Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand.

Science.gov (United States)

Teasley, Daniel C; Parajuli, Shankar; Nguyen, Mai; Moore, Hayley R; Alspach, Elise; Lock, Ying Jie; Honaker, Yuchi; Saharia, Abhishek; Piwnica-Worms, Helen; Stewart, Sheila A

2015-06-12

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Coincident resection at both ends of random, γ-induced double-strand breaks requires MRX (MRN, Sae2 (Ctp1, and Mre11-nuclease.

Directory of Open Access Journals (Sweden)

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

Directory of Open Access Journals (Sweden)

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.
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.

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
The DNA Repair Repertoire of Mycobacterium smegmatis FenA Includes the Incision of DNA 5' Flaps and the Removal of 5' Adenylylated Products of Aborted Nick Ligation.

Science.gov (United States)

Uson, Maria Loressa; Ghosh, Shreya; Shuman, Stewart

2017-09-01

We characterize Mycobacterium smegmatis FenA as a manganese-dependent 5'-flap endonuclease homologous to the 5'-exonuclease of DNA polymerase I. FenA incises a nicked 5' flap between the first and second nucleotides of the duplex segment to yield a 1-nucleotide gapped DNA, which is then further resected in dinucleotide steps. Initial FenA cleavage at a Y-flap or nick occurs between the first and second nucleotides of the duplex. However, when the template 3' single strand is eliminated to create a 5'-tailed duplex, FenA incision shifts to between the second and third nucleotides. A double-flap substrate with a mobile junction (mimicking limited strand displacement synthesis during gap repair) is preferentially incised as the 1-nucleotide 3'-flap isomer, with the scissile phosphodiester shifted by one nucleotide versus a static double flap. FenA efficiently removes the 5' App(dN) terminus of an aborted nick ligation reaction intermediate, thereby highlighting FenA as an agent of repair of such lesions, which are formed under a variety of circumstances by bacterial NAD + -dependent DNA ligases and especially by mycobacterial DNA ligases D and C. IMPORTANCE Structure-specific DNA endonucleases are implicated in bacterial DNA replication, repair, and recombination, yet there is scant knowledge of the roster and catalytic repertoire of such nucleases in Mycobacteria This study identifies M. smegmatis FenA as a stand-alone endonuclease homologous to the 5'-exonuclease domain of mycobacterial DNA polymerase 1. FenA incises 5' flaps, 5' nicks, and 5' App(dN) intermediates of aborted nick ligation. The isolated N-terminal domain of M. smegmatis Pol1 is also shown to be a flap endonuclease. Copyright © 2017 American Society for Microbiology.
Identification of human flap endonuclease 1 (FEN1) inhibitors using a machine learning based consensus virtual screening.

Science.gov (United States)

Deshmukh, Amit Laxmikant; Chandra, Sharat; Singh, Deependra Kumar; Siddiqi, Mohammad Imran; Banerjee, Dibyendu

2017-07-25

Human Flap endonuclease1 (FEN1) is an enzyme that is indispensable for DNA replication and repair processes and inhibition of its Flap cleavage activity results in increased cellular sensitivity to DNA damaging agents (cisplatin, temozolomide, MMS, etc.), with the potential to improve cancer prognosis. Reports of the high expression levels of FEN1 in several cancer cells support the idea that FEN1 inhibitors may target cancer cells with minimum side effects to normal cells. In this study, we used large publicly available, high-throughput screening data of small molecule compounds targeted against FEN1. Two machine learning algorithms, Support Vector Machine (SVM) and Random Forest (RF), were utilized to generate four classification models from huge PubChem bioassay data containing probable FEN1 inhibitors and non-inhibitors. We also investigated the influence of randomly selected Zinc-database compounds as negative data on the outcome of classification modelling. The results show that the SVM model with inactive compounds was superior to RF with Matthews's correlation coefficient (MCC) of 0.67 for the test set. A Maybridge database containing approximately 53 000 compounds was screened and top ranking 5 compounds were selected for enzyme and cell-based in vitro screening. The compound JFD00950 was identified as a novel FEN1 inhibitor with in vitro inhibition of flap cleavage activity as well as cytotoxic activity against a colon cancer cell line, DLD-1.
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
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.
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.
Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases

Directory of Open Access Journals (Sweden)

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.
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
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.
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...
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
Prospects for fen meadow restoration on severely degraded fens

NARCIS (Netherlands)

Klimkowska, Agata; Diggelen, Rudy Van; Grootjans, Ab P.; Kotowski, Wiktor

2010-01-01

The majority of fens in Europe have been transformed for agricultural purposes and have disappeared or become degraded. Fen meadows that developed under low-intensity management of fens also have become degraded. In this paper, we consider the available restoration methods, biotic constraints for
Recombinant Cyclophilins Lack Nuclease Activity

OpenAIRE

Manteca, Angel; Sanchez, Jesus

2004-01-01

Several single-domain prokaryotic and eukaryotic cyclophilins have been identified as also being unspecific nucleases with a role in DNA degradation during the lytic processes that accompany bacterial cell death and eukaryotic apoptosis. Evidence is provided here that the supposed nuclease activity of human and bacterial recombinant cyclophilins is due to contamination of the proteins by the host Escherichia coli endonuclease and is not an intrinsic property of these proteins.
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
[In vitro study of joint intervention of E-cad and Bmi-1 mediated by transcription activator-like effector nuclease in nasopharyngeal carcinoma].

Science.gov (United States)

Luo, Tingting; Yan, Aifen; Liu, Lian; Jiang, Hong; Feng, Cuilan; Liu, Guannan; Liu, Fang; Tang, Dongsheng; Zhou, Tianhong

2018-03-28

To explore the effect of intervention of E-cadherin (E-cad) and B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) mediated by transcription activator-like effector nuclease (TALEN) on the biological behaviors of nasopharyngeal carcinoma cells.  Methods: Multi-locus gene targeting vectors pUC-DS1-CMV-E-cad-2A-Neo-DS2 and pUC-DS1-Bmi-1 shRNA-Zeo-DS2 were constructed, and the E-cad and Bmi-1 targeting vectors were transferred with TALEN plasmids to CNE-2 cells individually or simultaneously. The integration of target genes were detected by PCR, the expressions of E-cad and Bmi-1 were detected by Western blot. The changes of cell proliferation were detected by cell counting kit-8 (CCK-8) assay. The cell cycle and apoptosis were detected by flow cytometry. The cell migration and invasion were detected by Transwell assay.  Results: The E-cad and Bmi-1 shRNA expression elements were successfully integrated into the genome of CNE-2 cells, the protein expression level of E-cad was up-regulated, and the protein expression level of Bmi-1 was down-regulated. The intervention of E-cad and Bmi-1 didn't affect the proliferation, cell cycle and apoptosis of CNE-2 cells, but it significantly inhibited the migration and invasion ability of CNE-2 cells. Furthermore, the intervention of E-cad and Bmi-1 together significantly inhibited the migration ability of nasopharyngeal carcinoma cells compared with the intervention of E-cad or Bmi-1 alone (all Pcad and Bmi-1 mediated by TALEN can effectively inhibit the migration and invasion of nasopharyngeal carcinoma cells in vitro, which may lay the preliminary experimental basis for gene therapy of human cancer.
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
Seed dispersal in fens

NARCIS (Netherlands)

Middleton, Beth; van Diggelen, Rudy; Jensen, Kai

Question: How does seed dispersal reduce fen isolation and contribute to biodiversity? Location: European and North American fens. Methods: This paper reviews the literature on seed dispersal to fens. Results: Landscape fragmentation may reduce dispersal opportunities thereby isolating fens and
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)
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.

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

KAUST Repository

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

2015-01-01

Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S.cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S.cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. © 2015 The Society for Biotechnology, Japan.
Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae

KAUST Repository

Aouida, Mustapha

2015-04-01

Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S.cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S.cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. © 2015 The Society for Biotechnology, Japan.
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
Characterization of Fen-Daqu Through Multivariate Statistical Analysis of H-1 NMR Spectroscopic Data

NARCIS (Netherlands)

Van-Diep, L.; Zheng, X.; Ma, K.; Chen, J.Y.; Han, B.Z.; Nout, M.J.R.

2011-01-01

J. Inst. Brew. 117(4), 516-522, 2011 Fen liquor is typical of Chinese light-flavour liquor (alcoholic spirit), which is fermented from sorghum with Fen-Daqu powder. Fen-Daqu is a saccharifying agent and fermentation starter in this fermentation process and in Fen traditional vinegar. To investigate
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.
Hydrological landscape settings of base-rich fen mires and fen meadows : an overview

NARCIS (Netherlands)

Grootjans, A.P.; Adema, E.B.; Bleuten, W.; Joosten, H.; Madaras, M.; Janáková, M.; Middleton, B.

2006-01-01

Question: Why do similar fen meadow communities occur in different landscapes? How does the hydrological system sustain base-rich fen mires and fen meadows? Location: Interdunal wetlands and heathland pools in The Netherlands, percolation mires in Germany, Poland, and Siberia, and calcareous spring
DNA Polymerase α (swi7) and the Flap Endonuclease Fen1 (rad2) Act Together in the S-Phase Alkylation Damage Response in S. pombe

Science.gov (United States)

Koulintchenko, Milana; Vengrova, Sonya; Eydmann, Trevor; Arumugam, Prakash; Dalgaard, Jacob Z.

2012-01-01

Polymerase α is an essential enzyme mainly mediating Okazaki fragment synthesis during lagging strand replication. A specific point mutation in Schizosaccharomyces pombe polymerase α named swi7-1, abolishes imprinting required for mating-type switching. Here we investigate whether this mutation confers any genome-wide defects. We show that the swi7-1 mutation renders cells hypersensitive to the DNA damaging agents methyl methansulfonate (MMS), hydroxyurea (HU) and UV and incapacitates activation of the intra-S checkpoint in response to DNA damage. In addition we show that, in the swi7-1 background, cells are characterized by an elevated level of repair foci and recombination, indicative of increased genetic instability. Furthermore, we detect novel Swi1-, -Swi3- and Pol α- dependent alkylation damage repair intermediates with mobility on 2D-gel that suggests presence of single-stranded regions. Genetic interaction studies showed that the flap endonuclease Fen1 works in the same pathway as Pol α in terms of alkylation damage response. Fen1 was also required for formation of alkylation- damage specific repair intermediates. We propose a model to explain how Pol α, Swi1, Swi3 and Fen1 might act together to detect and repair alkylation damage during S-phase. PMID:23071723
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.
Fens As Ecohydrologic Gauges of Climate Change in California

Science.gov (United States)

Drexler, J. Z.; Flint, L. E.; Flint, A. L.; Knifong, D. L.

2014-12-01

would likely lead to impacts on wildlife populations. The establishment of fen monitoring networks could be a highly effective method by which to identify and track both hydrologic and ecologic impacts from climate change in California and potentially other parts of the arid West.
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.
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
Human FEN1 Expression and Solubility Patterson in DNA Replication and Repair

National Research Council Canada - National Science Library

Carrier, Richard

1999-01-01

.... I show that human FEN1 mRNA and protein levels increase in a cell cycle-dependent manner, with peak mRNA and protein levels attained coincident with S phase DNA replication in both primary and transformed cells...
Geochemistry and hydrology of a calcareous fen within the Savage Fen wetlands complex, Minnesota, USA

Science.gov (United States)

Komor, S.C.

1994-01-01

Savage Fen is a wetlands complex at the base of north-facing bluffs in the Minnesota River Valley. The complex includes 27.8 hectares of calcareous fen that host rare calciphile plants whose populations are declining in Minnesota. Water and sediment compositions in the calcareous fen were studied to gain a better understanding of the hydrologie System that sustains the rare vegetation. Groundwater in the fen is a calcium-magnesium-bicarbonate type with circumneutral pH values. The groundwater composition is the resuit of interactions among water, dissolved and gaseous carbon species, carbonates, and ion exchangers. Shallow groundwater is distinguished from deep groundwater by smaller concentrations of chloride, sulfate, magnesium, and sodium, and larger concentrations of calcium, bicarbonate, hydrogen sulfide, and ammonium. Magnesian calcite is the prevalent carbonate in unconsolidated sedimentary fill beneath the fen and is an important source and sink for dissolved calcium, magnesium, and inorganic carbon. Calcite concentrations just below the water table are small because aerobic and anaerobic oxidation of organic matter increase the partial pressure of carbon dioxide (PCO2), decrease pH, and cause calcite to dissolve. Thick calcite accumulations just above the water table, in the root zone of calciphile plants, result from water table fluctuations and attendant changes in PCO2. Groundwater beneath Savage Fen recharges in lakes and ponds south of the fen and upwells to the surface within the fen. Water at the water table is a mixture of upwelling groundwater and water near the surface that flows downslope from higher elevations in the fen. Changes in oxygen and hydrogen isotope compositions of shallow groundwater indicate that the proportion of upwelling groundwater in shallow groundwater decreases downgradient in the calcareous fen. Encroachment of reed grasses into the calcareous fen may reflect human-caused disturbances in the recharge area.
Innovative wetland reclamation design case studies : the Suncor fen and pond 1 marsh

Energy Technology Data Exchange (ETDEWEB)

Daly, C.A. [Suncor Energy, Fort McMurray, AB (Canada); Price, J.; Rezanezhad, F. [Waterloo Univ., ON (Canada); Rochefort, L.; Graf, M. [Laval Univ., Quebec City, PQ (Canada); Russell, B. [BGC Engineering Inc., Calgary, AB (Canada)

2010-07-01

Suncor Energy Inc. is an energy company strategically focused in Canada's Athabasca oil sands region. The company has more than 40 years of experience in pioneering commercial development. This presentation discussed reclamation requirements for Suncor Energy. Reclamation objectives that were addressed in this presentation included operating approvals, biodiversity and meeting stakeholder expectations. Several photographs of reclaimed forest areas were shown along with 5 freshwater wetland types and wetland values. Two innovative wetland reclamation design case studies were also presented, described and illustrated, with particular reference to the Suncor fen and pond 1 marsh. It was concluded that future work will involve finalizing site investigation and designs, as well as construction, vegetation, and monitoring. figs.
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
Transplantation of Gene-Edited Hepatocyte-like Cells Modestly Improves Survival of Arginase-1-Deficient Mice

Directory of Open Access Journals (Sweden)

Yuan Yan Sin

2018-03-01

Full Text Available Progress in gene editing research has been accelerated by utilizing engineered nucleases in combination with induced pluripotent stem cell (iPSC technology. Here, we report transcription activator-like effector nuclease (TALEN-mediated reincorporation of Arg1 exons 7 and 8 in iPSCs derived from arginase-1-deficient mice possessing Arg1Δ alleles lacking these terminal exons. The edited cells could be induced to differentiate into hepatocyte-like cells (iHLCs in vitro and were subsequently used for transplantation into our previously described (Sin et al., PLoS ONE 2013 tamoxifen-inducible Arg1-Cre arginase-1-deficient mouse model. While successful gene-targeted repair was achieved in iPSCs containing Arg1Δ alleles, only minimal restoration of urea cycle function could be observed in the iHLC-transplanted mice compared to control mice, and survival in this lethal model was extended by up to a week in some mice. The partially rescued phenotype may be due to inadequate regenerative capacity of arginase-1-expressing cells in the correct metabolic zones. Technical hurdles exist and will need to be overcome for gene-edited iPSC to iHLC rescue of arginase-1 deficiency, a rare urea cycle disorder.
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
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
PRMT1 methylates the single Argonaute of Toxoplasma gondii and is important for the recruitment of Tudor nuclease for target RNA cleavage by antisense guide RNA

Science.gov (United States)

Musiyenko, Alla; Majumdar, Tanmay; Andrews, Joel; Adams, Brian; Barik, Sailen

2013-01-01

Summary Argonaute (Ago) plays a central role in RNA interference in metazoans, but its status in lower organisms remains ill-defined. We report on the Ago complex of the unicellular protozoan, Toxoplasma gondii (Tg), an obligatory pathogen of mammalian hosts. The PIWI-like domain of TgAgo lacked the canonical DDE/H catalytic triad, explaining its weak target RNA cleavage activity. However, TgAgo associated with a stronger RNA slicer, a Tudor staphylococcal nuclease (TSN), and with a protein Arg methyl transferase, PRMT1. Mutational analysis suggested that the N-terminal RGG-repeat domain of TgAgo was methylated by PRMT1, correlating with the recruitment of TSN. The slicer activity of TgAgo was Mg2+-dependent and required perfect complementarity between the guide RNA and the target. In contrast, the TSN activity was Ca2+-dependent and required an imperfectly paired guide RNA. Ago knockout parasites showed essentially normal growth, but in contrast, the PRMT1 knockouts grew abnormally. Chemical inhibition of Arg-methylation also had an anti-parasitic effect. These results suggest that the parasitic PRMT1 plays multiple roles, and its loss affects the recruitment of a more potent second slicer to the parasitic RNA silencing complex, the exact mechanism of which remains to be determined. PMID:22309152

Combinatorial regulation of meiotic holliday junction resolution in C. elegans by HIM-6 (BLM) helicase, SLX-4, and the SLX-1, MUS-81 and XPF-1 nucleases.

Science.gov (United States)

Agostinho, Ana; Meier, Bettina; Sonneville, Remi; Jagut, Marlène; Woglar, Alexander; Blow, Julian; Jantsch, Verena; Gartner, Anton

2013-01-01

Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants showed a similar reduction in survival rates as slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of "univalents" linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation
A cryptic targeting signal creates a mitochondrial FEN1 isoform with tailed R-Loop binding properties.

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Lawrence Kazak

Full Text Available A growing number of DNA transacting proteins is found in the nucleus and in mitochondria, including the DNA repair and replication protein Flap endonuclease 1, FEN1. Here we show a truncated FEN1 isoform is generated by alternative translation initiation, exposing a mitochondrial targeting signal. The shortened form of FEN1, which we term FENMIT, localizes to mitochondria, based on import into isolated organelles, immunocytochemistry and subcellular fractionation. In vitro FENMIT binds to flap structures containing a 5' RNA flap, and prefers such substrates to single-stranded RNA. FENMIT can also bind to R-loops, and to a lesser extent to D-loops. Exposing human cells to ethidium bromide results in the generation of RNA/DNA hybrids near the origin of mitochondrial DNA replication. FENMIT is recruited to the DNA under these conditions, and is released by RNase treatment. Moreover, high levels of recombinant FENMIT expression inhibit mtDNA replication, following ethidium bromide treatment. These findings suggest FENMIT interacts with RNA/DNA hybrids in mitochondrial DNA, such as those found at the origin of replication.
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.
Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.

Science.gov (United States)

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

2015-05-01

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle. © 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
Genetic correction using engineered nucleases for gene therapy applications.

Science.gov (United States)

Li, Hongmei Lisa; Nakano, Takao; Hotta, Akitsu

2014-01-01

Genetic mutations in humans are associated with congenital disorders and phenotypic traits. Gene therapy holds the promise to cure such genetic disorders, although it has suffered from several technical limitations for decades. Recent progress in gene editing technology using tailor-made nucleases, such as meganucleases (MNs), zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs) and, more recently, CRISPR/Cas9, has significantly broadened our ability to precisely modify target sites in the human genome. In this review, we summarize recent progress in gene correction approaches of the human genome, with a particular emphasis on the clinical applications of gene therapy. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.
Impacts of oil sands process water on fen plants: Implications for plant selection in required reclamation projects

International Nuclear Information System (INIS)

Pouliot, Rémy; Rochefort, Line; Graf, Martha D.

2012-01-01

Fen plant growth in peat contaminated with groundwater discharges of oil sands process water (OSPW) was assessed in a greenhouse over two growing seasons. Three treatments (non-diluted OSPW, diluted OSPW and rainwater) were tested on five vascular plants and four mosses. All vascular plants tested can grow in salinity and naphthenic acids levels currently produced by oil sands activity in northwestern Canada. No stress sign was observed after both seasons. Because of plant characteristics, Carex species (C. atherodes and C. utriculata) and Triglochin maritima would be more useful for rapidly restoring vegetation and creating a new peat-accumulating system. Groundwater discharge of OSPW proved detrimental to mosses under dry conditions and ensuring adequate water levels would be crucial in fen creation following oil sands exploitation. Campylium stellatum would be the best choice to grow in contaminated areas and Bryum pseudotriquetrum might be interesting as it has spontaneously regenerated in all treatments. - Highlights: ► Fen plant growth was assessed under groundwater discharges of oil sands process water. ► Sedge and grass species were not stressed after two growing seasons in greenhouse. ► Carex species and Triglochin maritima would be helpful in created contaminated fens. ► In dry conditions, contaminated groundwater discharge was detrimental for mosses. ► Campylium stellatum would be the best choice in created fens with contaminated water. - Sedges and grasses tolerated the contact with oil sands process water and could probably grow well in contaminated created fens, but mosses were particularly affected under dry conditions.
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...
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.
Effect of Groundwater Abstraction on Fen Ecosystems

DEFF Research Database (Denmark)

Johansen, Ole; Pedersen, Morten Lauge; Jensen, Jacob Birk

2011-01-01

within a distance of 1.5 km to a planned well field. In the river valley the interaction between groundwater and surface water is strongly affected by low permeable sediments. These sediments reduce the direct discharge to the river and have a large impact on the functioning and presence of the rich fen......Quantifying the effects of groundwater abstraction on fen ecosystems located in discharge areas can be complicated. The water level in fens is close to the terrain surface most of the year and it is controlled by a relatively constant groundwater exfiltration. It is difficult to measure...... the exfiltration fluxes and thus water level data is typically used to evaluate if the ecosystem is affected. The paper presents collected data and analysis from a case study, where the hydrological effect of groundwater abstraction on rich fens and springs in a Danish river valley has been studied. The natural...
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.
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
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.
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
DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction*

Science.gov (United States)

Algasaier, Sana I.; Exell, Jack C.; Bennet, Ian A.; Thompson, Mark J.; Gotham, Victoria J. B.; Shaw, Steven J.; Craggs, Timothy D.; Finger, L. David; Grasby, Jane A.

2016-01-01

Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr40, Asp181, and Arg100 and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage. PMID:26884332
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.
Zebrafish Model of NF1 for Structure-Function Analysis, Mechanisms of Glial Tumorigenesis, and Chemical Biology

Science.gov (United States)

2015-08-01

models. Zinc-finger nucleases (ZFNs) are chimeric fusions between a zinc-finger protein (ZFP) and the nuclease domain of FokI (Urnov et al., 2010). ZFNs...reference line. Once a target site is chosen, the user can click on the ZFN entry (QueryID; see Fig. 4B) for details about each construct (Fig. 4C...Zhong, Y. (1997). Requirement of Drosophila NF1 for activation of adenylyl cyclase by PACAP38-like neuro- peptides . Science 276, 795–798.ports 8, 1265
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
The hydrological functioning of a constructed fen wetland watershed.

Science.gov (United States)

Ketcheson, Scott J; Price, Jonathan S; Sutton, Owen; Sutherland, George; Kessel, Eric; Petrone, Richard M

2017-12-15

Mine reclamation requires the reconstruction of entire landforms and drainage systems. The hydrological regime of reclaimed landscapes will be a manifestation of the processes operating within the individual landforms that comprise it. Hydrology is the most important process regulating wetland function and development, via strong controls on chemical and biotic processes. Accordingly, this research addresses the growing and immediate need to understand the hydrological processes that operate within reconstructed landscapes following resource extraction. In this study, the function of a constructed fen watershed (the Nikanotee Fen watershed) is evaluated for the first two years following construction (2013-2014) and is assessed and discussed within the context of the construction-level design. The system design was capable of sustaining wet conditions within the Nikanotee Fen during the snow-free period in 2013 and 2014, with persistent ponded water in some areas. Evapotranspiration dominated the water fluxes from the system. These losses were partially offset by groundwater discharge from the upland aquifer, which demonstrated strong hydrologic connectivity with the fen in spite of most construction materials having lower than targeted saturated hydraulic conductivities. However, the variable surface infiltration rates and thick placement of a soil-capping layer constrained recharge to the upland aquifer, which remained below designed water contents in much of the upland. These findings indicate that it is possible to engineer the landscape to accommodate the hydrological functions of a fen peatland following surface oil sands extraction. Future research priorities should include understanding the storage and release of water within coarse-grained reclaimed landforms as well as evaluating the relative importance of external water sources and internal water conservation mechanisms for the viability of fen ecosystems over the longer-term. Copyright © 2017 Elsevier B
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
BOREAS TGB-1/TGB-3 NEE Data over the NSA Fen

Science.gov (United States)

Bellisario, Lianne; Hall, Forrest G. (Editor); Conrad, Sara K. (Editor); Moore, Tim R.

2000-01-01

The BOReal Ecosystem-Atmosphere Study Trace Gas Biogeochemistry (BOREAS TGB-1) and TGB-3 teams collected several data sets that contributed to understanding the measured trace gas fluxes over sites in the Northern Study Area (NSA). This data set contains Net Ecosystem Exchange of CO2 (NEE) measurements collected with chambers at the NSA fen in 1994 and 1996. Gas samples were extracted approximately every 7 days from chambers and analyzed at the NSA lab facility. The data are provided in tabular ASCII files.

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

Directory of Open Access Journals (Sweden)

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.
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.
Efficient methods for targeted mutagenesis in zebrafish using zinc-finger nucleases: data from targeting of nine genes using CompoZr or CoDA ZFNs.

Directory of Open Access Journals (Sweden)

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.
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
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
Structure features of TBN1, a P1/S1-like nuclease

Czech Academy of Sciences Publication Activity Database

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

2014-01-01

Roč. 281, Supplement s1 (2014), s. 601 ISSN 1742-464X. [FEBS EMBO 2014 Conference. 30.08.2014-04.09.2014, Paris] R&D Projects: GA MŠk(CZ) EE2.3.30.0029; GA MŠk LG14009; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61389013 ; RVO:86652036 ; RVO:60077344 Keywords : chitin * chitinase * clostridium Subject RIV: CE - Biochemistry; EI - Biotechnology ; Bionics (BTO-N); EB - Genetics ; Molecular Biology (BC-A) http://onlinelibrary.wiley.com/doi/10.1111/febs.12919/abstract
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.
Repair of DNA-polypeptide crosslinks by human excision nuclease

Science.gov (United States)

Reardon, Joyce T.; Sancar, Aziz

2006-03-01

DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome. damage recognition | nucleotide excision repair
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
Casein kinase 1-Like 3 is required for abscisic acid regulation of ...

African Journals Online (AJOL)

Casein kinase 1-Like 3 is required for abscisic acid regulation of seed germination, root growth, and gene expression in Arabidopsis. M Wang, D Yu, X Guo, X Li, J Zhang, L Zhao, H Chang, S Hu, C Zhang, J Shi, X Liu ...
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.
Cell wall-anchored nuclease of Streptococcus sanguinis contributes to escape from neutrophil extracellular trap-mediated bacteriocidal activity.

Directory of Open Access Journals (Sweden)

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.
Unravelling hydrological mechanisms behind fen deterioration

NARCIS (Netherlands)

van Loon, A.H.

2010-01-01

Low-productive fens are wetland ecosystems that have strongly declined in number and size in temperate regions of Western Europe since World War II. This fen type has significant conservation value in many countries because of its high species diversity and the presence of endangered plant species.
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.
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...
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
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.
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.
The distribution and migration of sodium from a reclaimed upland to a constructed fen peatland in a post-mined oil sands landscape.

Science.gov (United States)

Kessel, Eric D; Ketcheson, Scott J; Price, Jonathan S

2018-07-15

Post-mine landscape reclamation of the Athabasca Oil Sands Region requires the use of tailings sand, an abundant mine-waste material that often contains large amounts of sodium (Na + ). Due to the mobility of Na + in groundwater and its effects on vegetation, water quality is a concern when incorporating mine waste materials, especially when attempting to construct groundwater-fed peatlands. This research is the first published account of Na + redistribution in groundwater from a constructed tailings sand upland to an adjacent constructed fen peat deposit (Nikanotee Fen). A permeable petroleum coke layer underlying the fen, extending partway into the upland, was important in directing flow and Na + beneath the peat, as designed. Initially, Na + concentration was highest in the tailings sand (average of 232mgL -1 ) and lowest in fen peat (96mgL -1 ). Precipitation-driven recharge to the upland controlled the mass flux of Na from upland to fen, which ranged from 2 to 13tons Na + per year. The mass flux was highest in the driest summer, in part from dry-period flowpaths that direct groundwater with higher concentrations of Na + into the coke layer, and in part because of the high evapotranspiration loss from the fen in dry periods, which induces upward water flow. With the estimated flux rates of 336mmyr -1 , the Na + arrival time to the fen surface was estimated to be between 4 and 11years. Over the four-year study, average Na + concentrations within the fen rooting zone increased from 87 to 200mgL -1 , and in the tailings sand decreased to 196mgL -1 . The planting of more salt-tolerant vegetation in the fen is recommended, given the potential for Na + accumulation. This study shows reclamation designs can use layered flow system to control the rate, pattern, and timing of solute interactions with surface soil systems. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases.

Directory of Open Access Journals (Sweden)

Craig B Wilen

2011-04-01

Full Text Available HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5 virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4 in place of or in addition to CCR5 (R5X4 remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.
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.
Experimental restoration of a fen plant community after peat mining

Energy Technology Data Exchange (ETDEWEB)

Cobbaert, D.; Rochefort, L.; Price, J.S. [Univ. Laval, Sainte-Foy (Canada). Dept. de Phytologie

2004-11-01

Methods: The effectiveness of introducing fen plants with the application of donor diaspore material was tested. The donor diaspore material, containing seeds, rhizomes, moss fragments, and other plant propagules, was collected from two different types of natural fens. We tested whether the application of straw mulch would increase fen species cover and biodiversity compared to control plots without straw mulch. Terrace levels of different peat depths (15 cm, 40 cm, and 56 cm) were created to test the effects of different environmental site conditions on the success of re-vegetation. Results: Applying donor seed bank from natural fens was found to significantly increase fen plant cover and richness after the two growing seasons. Straw mulch proved to significantly increase fen plant richness. The intermediate terrace level (40 cm) had the highest fen plant establishment. Compared to reference sites, the low terrace level (15 cm) was richer in base cations, whereas the high terrace level (56 cm) was much drier. Conclusions: The application of donor diaspore material was demonstrated as an effective technique for establishing vascular fen plants. Further rewetting measures are considered necessary at the restoration site to create a fen ecosystem rather than simply restoring some fen species (Location: Riviere-du-Loup peatland, southern Quebec, Canada at 100 m a.s.l.)
Targeted gene disruption by use of transcription activator-like effector nuclease (TALEN) in the water flea Daphnia pulex.

Science.gov (United States)

Hiruta, Chizue; Ogino, Yukiko; Sakuma, Tetsushi; Toyota, Kenji; Miyagawa, Shinichi; Yamamoto, Takashi; Iguchi, Taisen

2014-11-18

The cosmopolitan microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have its complete genome sequenced, an unprecedented ca. 36% of which has no known homologs with any other species. Moreover, D. pulex is ideally suited for experimental manipulation because of its short reproductive cycle, large numbers of offspring, synchronization of oocyte maturation, and other life history characteristics. However, existing gene manipulation techniques are insufficient to accurately define gene functions. Although our previous investigations developed an RNA interference (RNAi) system in D. pulex, the possible time period of functional analysis was limited because the effectiveness of RNAi is transient. Thus, in this study, we developed a genome editing system for D. pulex by first microinjecting transcription activator-like effector nuclease (TALEN) mRNAs into early embryos and then evaluating TALEN activity and mutation phenotypes. We assembled a TALEN construct specific to the Distal-less gene (Dll), which is a homeobox transcription factor essential for distal limb development in invertebrates and vertebrates, and evaluated its activity in vitro by single-strand annealing assay. Then, we injected TALEN mRNAs into eggs within 1 hour post-ovulation. Injected embryos presented with defects in the second antenna and altered appendage development, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene. We succeeded, for the first time in D. pulex, in targeted mutagenesis by use of Platinum TALENs. This genome editing technique makes it possible to conduct reverse genetic analysis in D. pulex, making this species an even more appropriate model organism for environmental, evolutionary, and developmental genomics.
TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells

DEFF Research Database (Denmark)

Pedersen, Rune Troelsgaard; Kruse, Thomas; Nilsson, Jakob

2015-01-01

mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise...... temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin...
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.
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.
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.
Synthesis, characterisation, nuclease and cytotoxic activity of ...

Indian Academy of Sciences (India)

GULZAR A BHAT

2018-02-07

Feb 7, 2018 ... 2 were evaluated for their nuclease and in vitro anti-tumor activities against human breast and colorectal cancer cell lines. The DNA ... tive chemotherapeutic agent for the treatment of ovarian, lung, testicular, colon, and neck ... coma, leukemia, Hodgkin's lymphoma, brain tumours and cancer of the cervix, ...
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
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.
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....
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.
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
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. ∗.
Plant Ribonucleases and Nucleases as Antiproliferative Agens Targeting Human Tumors Growing in Mice

Czech Academy of Sciences Publication Activity Database

Matoušek, Jaroslav; Matoušek, Josef

2010-01-01

Roč. 4, č. 1 (2010), s. 29-39 ISSN 1872-2156 R&D Projects: GA ČR GA521/06/1149; GA ČR GA521/09/1214 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z50450515 Keywords : antiproliferative cytotoxic * effect human * plant nuclease Subject RIV: EB - Genetics ; Molecular Biology
Interactions between the APOA5 -1131T>C and the FEN1 10154G>T polymorphisms on ω6 polyunsaturated fatty acids in serum phospholipids and coronary artery disease

Science.gov (United States)

Park, Ju Yeon; Paik, Jean Kyung; Kim, Oh Yoen; Chae, Jey Sook; Jang, Yangsoo; Lee, Jong Ho

2010-01-01

We determined the contribution of the combination of FEN1 10154G>T with the most significant association in the analysis of plasma arachidonic acid (AA, 20:4ω6) and the APOA5-1131T>C on phospholipid ω6PUFA and coronary artery disease (CAD). Patients with CAD (n = 807, 27–81 years of age) and healthy controls (n = 1123) were genotyped for FEN1 10154G>T and APOA5-1131T>C. We found a significant interaction between these two genes for CAD risk (P = 0.007) adjusted for confounding factors. APOA5-1131C allele carriers had a higher CAD risk [odds ratio (OR):1.484, 95% confidence interval (CI):1.31–1.96; P = 0.005] compared with APOA5-1131TT individuals in the FEN1 10154GG genotype group but not in the FEN1 10154T allele group (OR:1.096, 95%CI:0.84–1.43; P = 0.504). Significant interactions between these two genes were also observed for the AA proportion (P = 0.04) and the ratio of AA/linoleic acid (LA, 18:2ω6) (P = 0.004) in serum phospholipids of controls. The APOA5-1131C allele was associated with lower AA (P = 0.027) and AA/LA (P = 0.014) only in controls carrying the FEN1 10154T allele. In conclusion, the interaction between these genes suggests that the FEN1 10154T variant allele decreases AA and AA/LA in the serum phospholipids of carriers of the APOA5-1131C allele, but contributes no significant increase in CAD risk for this population subset despite their increased triglylcerides and decreased apoA5. PMID:20802161
Fen (n=1–6) clusters chemisorbed on vacancy defects in graphene: Stability, spin-dipole moment, and magnetic anisotropy

KAUST Repository

Haldar, Soumyajyoti; Pujari, Bhalchandra S.; Bhandary, Sumanta; Cossu, Fabrizio; Eriksson, Olle; Kanhere, Dilip G.; Sanyal, Biplab

2014-01-01

In this work, we have studied the chemical and magnetic interactions of Fen (n=1–6) clusters with vacancy defects (monovacancy to correlated vacancies with six missing C atoms) in a graphene sheet by ab initio density functional calculations
Compostos fenólicos e capacidade antioxidante em frutos de tomateiros mutantes fotomorfogenéticos

Directory of Open Access Journals (Sweden)

Tiago Dias

2015-05-01

Full Text Available Compostos fenólicos e capacidade antioxidante são mecanismos de defesa das plantas aos danos do estresse oxidativo. Os compostos fenólicos são sintetizados pela via dos fenilpropanoides, cuja enzima chave, fenilalanina amônia liase, é influenciada pela luz e ação de fotorreceptores, como o fitocromo. O objetivo do presente trabalho é avaliar a concentração de compostos fenólicos e a capacidade antioxidante de frutos de microtomateiro selvagem, cultivar "Micro-Tom" (MT, e seus mutantes fotomorfogenéticos high pigment 1 (hp1, super-responsivo a eventos mediados por luz e aurea (au, deficiente quantitativo em fitocromos. Vinte frutos maduros de cada genótipo (MT, hp1 e au foram utilizados para as análises, realizadas em triplicata. Para quantificação dos compostos fenólicos totais, foi utilizado o método de Folin-Ciocalteu e a capacidade antioxidante foi realizada pelos métodos Ferric Reducing Antioxidant Power (FRAP e 2,2-diphenyl-1-picrylhydrazyl (DPPH. Os frutos do mutante hp1 apresentaram maiores conteúdos de compostos fenólicos totais e também maior capacidade antioxidante em relação à cultivar selvagem ("MT" e ao mutante au, o qual não diferiu significativamente da cultivar "MT".
26 CFR 301.6036-1 - Notice required of executor or of receiver or other like fiduciary.

Science.gov (United States)

2010-04-01

... 26 Internal Revenue 18 2010-04-01 2010-04-01 false Notice required of executor or of receiver or... and Returns Returns and Records § 301.6036-1 Notice required of executor or of receiver or other like.... (b) Executors, administrators, and persons in possession of property of decedent. For provisions...

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...
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
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.
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
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.
El concepto de fenómeno en el joven Heidegger

Directory of Open Access Journals (Sweden)

Francisco De Lara

2008-01-01

Full Text Available El presente trabajo es una discusión del concepto de fenómeno tal y como éste se encuentra determinado en la filosofía del joven Heidegger. Con ella se pretende ir a la base que permite dilucidar en qué sentido hace éste fenomenología o, en otras palabras, qué idea de filosofía se encuentra expresada bajo el título de hermenéutica fenomenológica. Al hilo de la lectura del concepto de fenómeno como concepto filosófico, esto es, como indicación formal, nos preguntaremos: 1 cómo se toma en consideración lo investigado fenomenológicamente; 2 cómo debe comparecer lo así investigado y 3 si, en efecto, el concepto de fenómeno funciona siempre en Heidegger como una indicación formal o si no puede constatarse en él una cierta ambigüedad.
Responses of fen plant species to groundwater level and light intensity

NARCIS (Netherlands)

Kotowski, W; van Andel, J; van Diggelen, R; Hogendorf, J.

Characteristic species of sedge-moss fen communities occur in constantly wet, nutrient-poor sites with a high penetration of light through the vegetation canopy. We studied the effects of water table depth and differences in light intensity on the performance of fen species. Three fen species (Carex
Understanding the Groundwater Hydrology of a Geographically-Isolated Prairie Fen: Implications for Conservation.

Science.gov (United States)

Sampath, Prasanna Venkatesh; Liao, Hua-Sheng; Curtis, Zachary Kristopher; Doran, Patrick J; Herbert, Matthew E; May, Christopher A; Li, Shu-Guang

2015-01-01

The sources of water and corresponding delivery mechanisms to groundwater-fed fens are not well understood due to the multi-scale geo-morphologic variability of the glacial landscape in which they occur. This lack of understanding limits the ability to effectively conserve these systems and the ecosystem services they provide, including biodiversity and water provisioning. While fens tend to occur in clusters around regional groundwater mounds, Ives Road Fen in southern Michigan is an example of a geographically-isolated fen. In this paper, we apply a multi-scale groundwater modeling approach to understand the groundwater sources for Ives Road fen. We apply Transition Probability geo-statistics on more than 3000 well logs from a state-wide water well database to characterize the complex geology using conditional simulations. We subsequently implement a 3-dimensional reverse particle tracking to delineate groundwater contribution areas to the fen. The fen receives water from multiple sources: local recharge, regional recharge from an extensive till plain, a regional groundwater mound, and a nearby pond. The regional sources deliver water through a tortuous, 3-dimensional "pipeline" consisting of a confined aquifer lying beneath an extensive clay layer. Water in this pipeline reaches the fen by upwelling through openings in the clay layer. The pipeline connects the geographically-isolated fen to the same regional mound that provides water to other fen clusters in southern Michigan. The major implication of these findings is that fen conservation efforts must be expanded from focusing on individual fens and their immediate surroundings, to studying the much larger and inter-connected hydrologic network that sustains multiple fens.
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
RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency.

Science.gov (United States)

Song, Jun; Yang, Dongshan; Xu, Jie; Zhu, Tianqing; Chen, Y Eugene; Zhang, Jifeng

2016-01-28

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells.
RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency

Science.gov (United States)

Song, Jun; Yang, Dongshan; Xu, Jie; Zhu, Tianqing; Chen, Y. Eugene; Zhang, Jifeng

2016-01-01

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells. PMID:26817820
Calcareous Fens - Source Feature Points

Data.gov (United States)

Minnesota Department of Natural Resources — Pursuant to the provisions of Minnesota Statutes, section 103G.223, this database contains points that represent calcareous fens as defined in Minnesota Rules, part...
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.
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.
Actividad antioxidante de los compuestos fenólicos

Directory of Open Access Journals (Sweden)

Maestro Durán, R.

1993-04-01

Full Text Available This review deals with the oxidation processes induced by light and atmospheric oxygen, as well as the protective action on antioxidant compounds. The available data on the synthetic antioxidants and the problems associated to their use, together with the presence of natural phenolic antioxidants in plants, have been revised. These phenolic antioxidants have been classified in families: phenolic alcohols and acids, cinnamic acids, coumarins, favonoids (flavones and isoflavones, flavonols, flavanones and other phenolic compounds.

Este trabajo recoge los estudios sobre los mecanismos de oxidación producida por la luz y el oxígeno del aire y sobre la protección de los compuestos antioxidantes contra esta oxidación. Se han revisado los datos existentes sobre los inconvenientes de los antioxidantes sintéticos, que aconsejan sustituirlos por otros naturales y sobre la presencia de antioxidantes naturales de carácter fenólico en las plantas. Los antioxidantes fenólicos se han agrupado por familias: alcoholes y ácidos fenólicos, ácidos cinámicos, cumarinas, flavonoides (flavonas e isoflavonas, flavonoles, flavanonas y otros compuestos fenólicos.
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.
Determination of the 54Fe(n, 2n)53gFe and 54Fe(n, 2n)53mFe cross sections averaged over a 235U fission neutron spectrum

International Nuclear Information System (INIS)

Ribeiro Guevara, S.; Arribere, M.; Kestelman, A.J.

2002-01-01

The reaction cross sections averaged over a 235 U fission neutron spectrum have been measured for the 54 Fe(n, 2n) 53g Fe and 54 Fe(n, 2n) 53m Fe threshold reactions. The values found are, respectively: (1.14 ± 0.13) μb, and (0.52 ± 0.16) μb. The measured cross sections are referred to the (111± 3) mb standard cross section of the 58 Ni(n, p) 58m+g Co reaction. The (81.7 ± 2.2) mb standard cross section value for the 54 Fe(n, p) 54 Mn reaction, was also used as a monitor to check the results obtained with the Ni standard, leading to an excellent agreement. (author)
Fens and their rare plants in the Beartooth Mountains, Shoshone National Forest, Wyoming

Science.gov (United States)

Bonnie Heidel; Walter Fertig; Sabine Mellmann-Brown; Kent E. Houston; Kathleen A. Dwire

2017-01-01

Fens are common wetlands in the Beartooth Mountains on the Shoshone National Forest, Clarks Fork Ranger District, in Park County, Wyoming. Fens harbor plant species found in no other habitats, and some rare plants occurring in Beartooth fens are found nowhere else in Wyoming. This report summarizes the studies on Beartooth fens from 1962 to 2009, which have contributed...
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

Cofactor requirement of HpyAV restriction endonuclease.

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Siu-Hong Chan

Full Text Available BACKGROUND: Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M systems in microorganisms. PRINCIPAL FINDINGS: We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg(++. The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. CONCLUSIONS/SIGNIFICANCE: Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.
Establishment of pten knockout medaka with transcription activator-like effector nucleases (TALENs as a model of PTEN deficiency disease.

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Yuriko Matsuzaki

Full Text Available Phosphatase and tensin homolog (PTEN is a lipid and protein phosphatase that antagonizes signaling by the phosphatidylinositol 3-kinase (PI3K-AKT signaling pathway. The PTEN gene is a major tumor suppressor, with mutations of this gene occurring frequently in tumors of humans and mice. We have now developed mutant medaka deficient in PTEN with the use of transcription activator-like effector nuclease (TALEN technology. Medaka possesses two pten genes, ptena and ptenb, similar to zebrafish. We established 16 ptena mutant lines and two ptenb mutant lines. Homozygous single pten mutants were found to be viable and fertile. In contrast, pten double-knockout (dko embryos manifested severe abnormalities in vasculogenesis, eye size, and tail development at 72 hours post fertilization(hpf and died before hatching. Immunoblot analysis revealed that the ratio of phosphorylated to total forms of AKT (pAKT/AKT in pten dko embryos was four times that in wild-type embryos, indicative of up-regulation of signaling by the PI3K-AKT pathway. Treatment of pten dko embryos with the PI3K inhibitor LY294002 reduced the pAKT/AKT ratio by about one-half and partially rescued the defect in vasculogenesis. Additional inhibitors of the PI3K-AKT pathway, including rapamycin and N-α-tosyl-L-phenylalanyl chloromethyl ketone, also partially restored vasculogenesis in the dko embryos. Our model system thus allows pten dko embryos to be readily distinguished from wild-type embryos at an early stage of development and is suitable for the screening of drugs able to compensate for PTEN deficiency.
Formalized classification of European fen vegetation at the alliance level

DEFF Research Database (Denmark)

Peterka, Tomáš; Hájek, Michal; Jiroušek, Martin

2017-01-01

Aims Phytosociological classification of fen vegetation (Scheuchzerio palustris-Caricetea fuscae class) differs among European countries. Here we propose a unified vegetation classification of European fens at the alliance level, provide unequivocal assignment rules for individual vegetation plot...
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.
DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair.

Science.gov (United States)

de Laat, W L; Appeldoorn, E; Sugasawa, K; Weterings, E; Jaspers, N G; Hoeijmakers, J H

1998-08-15

The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5' side of its binding region, a weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning of the excision repair nucleases XPG and ERCC1-XPF on the DNA. With the 3'-oriented side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1-XPF, whereas the 5'-oriented side of hRPA at a DNA junction allows stable binding of XPG to hRPA. Our data pinpoint hRPA to the undamaged strand during nucleotide excision repair. Polarity of hRPA on ssDNA is likely to contribute to the directionality of other hRPA-dependent processes as well.
Glycoengineering of Human Cell Lines Using Zinc Finger Nuclease Gene Targeting

DEFF Research Database (Denmark)

Steentoft, Catharina; Bennett, Eric Paul; Clausen, Henrik

2013-01-01

Lectin affinity chromatography is a powerful technique for isolation of glycoproteins carrying a specific glycan structure of interest. However, the enormous diversity of glycans present on the cell surface, as well as on individual proteins, makes it difficult to isolate an entire glycoproteome...... with one or even a series of lectins. Here we present a technique to generate cell lines with homogenous truncated O-glycans using zinc finger nuclease gene targeting. Because of their simplified O-glycoproteome, the cells have been named SimpleCells. Glycoproteins from SimpleCells can be isolated...... in a single purification step by lectin chromatography performed on a long lectin column. This protocol describes Zinc finger nuclease gene targeting of human cells to simplify the glycoproteome, as well as lectin chromatography and isolation of glycopeptides from total cell lysates of SimpleCells....
Tsetse salivary gland proteins 1 and 2 are high affinity nucleic acid binding proteins with residual nuclease activity.

Directory of Open Access Journals (Sweden)

Guy Caljon

Full Text Available Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2 display DNA/RNA non-specific, high affinity nucleic acid binding with K(D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.
BOREAS TGB-1/TGB-3 CH4 Chamber Flux Data over the NSA Fen

Science.gov (United States)

Bubier, Jill L.; Moore, Tim R.; Hall, Forrest G. (Editor); Conrad, Sara K. (Editor)

2000-01-01

The BOREAS TGB-3 team collected methane (CH4) chamber flux measurements at the NSA fen site during May-September 1994 and June-October 1996. Gas samples were extracted approximately every 7 days from chambers and analyzed at the NSA lab facility. The data are provided in tabular ASCII files.
Three-dimensional information extraction from GaoFen-1 satellite images for landslide monitoring

Science.gov (United States)

Wang, Shixin; Yang, Baolin; Zhou, Yi; Wang, Futao; Zhang, Rui; Zhao, Qing

2018-05-01

To more efficiently use GaoFen-1 (GF-1) satellite images for landslide emergency monitoring, a Digital Surface Model (DSM) can be generated from GF-1 across-track stereo image pairs to build a terrain dataset. This study proposes a landslide 3D information extraction method based on the terrain changes of slope objects. The slope objects are mergences of segmented image objects which have similar aspects; and the terrain changes are calculated from the post-disaster Digital Elevation Model (DEM) from GF-1 and the pre-disaster DEM from GDEM V2. A high mountain landslide that occurred in Wenchuan County, Sichuan Province is used to conduct a 3D information extraction test. The extracted total area of the landslide is 22.58 ha; the displaced earth volume is 652,100 m3; and the average sliding direction is 263.83°. The accuracies of them are 0.89, 0.87 and 0.95, respectively. Thus, the proposed method expands the application of GF-1 satellite images to the field of landslide emergency monitoring.
Molecular characterization of shrimp harbinger transposase derived 1 (HARBI1)-like and its role in white spot syndrome virus and Vibrio alginolyticus infection.

Science.gov (United States)

Sun, Baozhen; Qian, Xiyi; Zhu, Fei

2018-07-01

The role of the nuclease, HARBI1-like protein (mjHARBI1-like) in the innate immunity of Marsupenaeus japonicus was explored in this study. The 1361 bp cDNA sequence of mjHARBI1-like was cloned from M. japonicus using RACE. RT-qPCR analysis results showed that the gills and hepatopancreas of M. japonicus were the main tissues where mjHARBI1-like is expressed. In addition, it was also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge could stimulate mjHARBI1-like expression. After mjHARBI1-likewas inhibited, expression of immune genes such as toll, p53, myosin, and proPO were significantly downregulated (P shrimp hemocytes, hemocyanin and tumor necrosis factor-α (TNF-α) were up-regulated significantly (P shrimp was significantly advanced by double-strand RNA interference (dsRNAi) of mjHARBI1-like. Apoptosis studies indicated that mjHARBI1-dsRNA treatment caused a reduction in hemocyte apoptosis in bacterial and viral groups. In addition, phagocytosis experiments illustrated that mjHARBI1-dsRNA treatment led to a lower phagocytosis rate in hemocytes of V. alginolyticus-challenged shrimp. It was also found that knockdown of mjHARBI1-like inhibited shrimp phenoloxidase (PO) activity, superoxide dismutase (SOD) activity, and total hemocyte count (THC) after WSSV or V. alginolyticus infection. These data indicate a regulative role of mjHARBI1-likein the immunity of shrimp in response to pathogen infection. Resultantly, it was concluded that mjHARBI1-like might have a positive effect on the anti-WSSV immune response of shrimp by regulating apoptosis, THC, PO activity, and SOD activity. Additionally, mjHARBI1-like might promote anti-V. alginolyticus infection by participating in regulating phagocytosis, apoptosis, SOD activity, PO activity, and THC. Copyright © 2018 Elsevier Ltd. All rights reserved.
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
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...
El fenómeno de envejecimiento de los asfaltos

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Xiomara Vargas

2010-09-01

Full Text Available En este trabajo se presenta una revisión actualizada a los resultados del estudio de los procesos de envejecimiento de los asfaltos, encontrados por diversos investigadores, con el fin de profundizar en la comprensión de este fenómeno complejo. Los antecedentes demuestran la evolución del estudio del fenómeno de envejecimiento de los asfaltos, considerado inicialmente como un proceso de endurecimiento físico y progresivamente reconocido como un fenómeno complejo con repercusiones en la durabilidad y las propiedades fisicoquímicas del asfalto. Este documento pretende ser una guía para el emprendimiento de futuras investigaciones relacionadas con el desarrollo de nuevos asfaltos y mezclas asfálticas con propiedades mejoradas.
Compostos fenólicos totais e antocianinas em suco de uva

OpenAIRE

Malacrida,Cassia R.; Motta,Silvana da

2005-01-01

As concentrações de compostos fenólicos totais e antocianinas em sucos de uva reconstituídos e simples, de diferentes marcas, disponíveis no comércio varejista da região metropolitana de Belo Horizonte (Minas Gerais) foram quantificadas. Também se determinou a contribuição das antocianinas poliméricas à cor e o índice de degradação das antocianinas. Os teores de compostos fenólicos totais variaram entre 0,27 e 2,41 g/L e as concentrações de antocianinas de 1,17 a 66,80 mg/L. Foram observadas ...
Stand structure and vegetation dynamics of a subalpine treed fen in Rocky Mountain National Park, Colorado

Energy Technology Data Exchange (ETDEWEB)

Johnson, J.B. [Colorado State Univ., Fort Collins, CO (United States). Dept. of Biology

1997-06-01

The tree population size structure and relationship between tree diameter and age were examined in a subalpine fen and surrounding Picea-Abies forest in northern Colorado. The fen grades from a sedge fen, through an ecotone, to a treed fen (i.e. fen colonized by trees). Tree growth rate varies across the vegetational gradient, with the sedge fen having the slowest growth, and the upland forest having the fastest growth. Differences in growth rate are related to the average size of peat hummocks, with areas containing tall hummocks exhibiting the highest tree growth rates. Size structures display the characteristic reverse-J distribution generally indicative of stable populations, but forest vegetation is expanding into the open regions of the fen, and within the treed fen an increase in Abies lasiocarpa is occurring. These changes are primarily attributed to a positive feedback situation wherein the fen`s surface is built up by peat accumulation. Distinct hummocks form first on the open fen but then coalesce to form raised peat islands in the treed fen. This new substrate provides habitat with a comparatively low water table and allows the growth of mesophytic forest vegetation. A pathway for this vegetational development is proposed. 40 refs., 2 figs.
Characterization of volatile compounds in Fen-Daqu - a traditional Chinese liquor fermentation starter

NARCIS (Netherlands)

Van-Diep, L.; Zheng, X.; Chen, J.Y.; Han, B.Z.

2012-01-01

Fen-Daqu is a saccharifying agent and fermentation starter for the production of Chinese liquor Fen (alcoholic spirit) and Fen traditional vinegar. The volatile compounds produced at seven incubation steps were analysed by HS-SPME-GC-MS. A total of 83 major volatile compounds were identified,
Formation dynamics of FeN thin films on Cu(100)

KAUST Repository

Heryadi, Dodi

2012-01-01

To investigate the structural and magnetic properties of thin films of FeN we have performed ab initio molecular dynamics simulations of their formation on Cu(100) substrates. The iron nitride layers exhibit a p4gm(2 × 2) reconstruction and order ferromagnetically in agreement with experiment. We establish the dynamics and time scale of the film formation as a function of the film thickness. The process is split in two phases: formation of almost flat FeN layers and optimization of the distance to the substrate. Our calculated magnetic moments are 1.67 μ B, 2.14 μ B, and 2.21 μ B for one, two, and three monolayers of iron nitride. © 2011 Elsevier B.V. All rights reserved.
Novel fen ecosystems in western North Carolina

Science.gov (United States)

Wilcox, J. D.

2012-12-01

Western North Carolina is mountainous, and groundwater flows from hillslope recharge zones to valley stream and spring discharge zones. Depending on surface topography and geologic conditions, the water table may approach or intersect the ground surface to form seepage wetlands, or fens. Fen ecosystems can be very sensitive to changes in land use, groundwater pumping, and upslope development. This presentation will focus on two sites where historical land use and human activity played important roles in creating or preserving fen ecosystems. Both sites now support—and are being managed to protect—federally endangered flora and fauna. The first site is home to Sarracenia oreophilia, an endangered pitcher plant that thrives on saturated soils with low nutrient content. The site's early history includes tree clearing, drain tile installation, and cattle grazing, while more recent management activities have included drain tile excavation, manual invasive removal, and prescribed burns. A 15-year water-level record indicates seasonal artesian conditions wet a 3m clay unit (K=2E-5 cm/sec) beneath the site, which is able to retain moisture during drier periods. Shorter "clay wetting periods" during drought years (1999-2000; 2007-2008) correspond to reduced clump counts in pitcher-plant surveys. The second site is a former aggregate quarry that now supports over 60 bog turtles (Clemmys muhlenbergii). The biggest threat to this site is encroachment of non-native and invasive multiflora rose (Rosa multiflora) and other large woody species. Management activities include manual removal and prescribed goat herbivory. Current efforts to characterize the springs, water-table, and surface-water flows will be used to detect changes in the future to the hydrologic regime in the fen.
Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein–staphylococcal nuclease hybrid

International Nuclear Information System (INIS)

Mori, Tomoaki; Nakamura, Kento; Masaoka, Keisuke; Fujita, Yusuke; Morisada, Ryosuke; Mori, Koichi; Tobimatsu, Takamasa; Sera, Takashi

2016-01-01

Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms. - Highlights: • A novel RNA restriction enzyme using SNase was developed tor cleave viral RNA. • Our enzyme cleaved influenza RNA with rates >120-fold higher rates a PIN-fusion one. • Our artificial enzyme with the L5 linker showed the highest RNA cleavage rate. • Our artificial enzyme site-selectively cleaved influenza RNA in vitro.
Modeling thermodynamics of Fe-N phases; characterisation of e-Fe2N1-z

DEFF Research Database (Denmark)

Pekelharing, M.I.; Böttger, A.; Somers, Marcel A.J.

1999-01-01

In order to arrive at modeling the thermodynamics of Fe-N phases, including long-range (LRO) and short-range ordering (SRO) of the N atoms, it is important to understand the role of N interstitially dissolved in an Fe-host lattice. The crystal structure of -Fe2N1-z consists of an h.c.p. iron...... sublattice and a hexagonal nitrogen sublattice formed by octahedral interstices of the Fe sublattice [1]. Two ground-state structures have been proposed for the ordered arrangement of the N atoms on their own sublattice [1], which were shown to be thermodynamically favourable [2]: configuration A for Fe2N1...... investigated with X-ray diffraction (XRD) and Mössbauer spectroscopy. A thermodynamic model accounting for the two configurations of LRO of the N atoms [2,3] was fitted to the N-absorption isotherm at 723 K and resulted in the occupancies of the sites of the nitrogen sublattice. A miscibility gap between...

Zinc finger nuclease: a new approach for excising HIV-1 proviral DNA from infected human T cells.

Science.gov (United States)

Qu, Xiying; Wang, Pengfei; Ding, Donglin; Wang, Xiaohui; Zhang, Gongmin; Zhou, Xin; Liu, Lin; Zhu, Xiaoli; Zeng, Hanxian; Zhu, Huanzhang

2014-09-01

A major reason that Acquired Immune Deficiency Syndrome (AIDS) cannot be completely cured is the human immunodeficiency virus 1 (HIV-1) provirus integrated into the human genome. Though existing therapies can inhibit replication of HIV-1, they cannot eradicate it. A molecular therapy gains popularity due to its specifically targeting to HIV-1 infected cells and effectively removing the HIV-1, regardless of viral genes being active or dormant. Now, we propose a new method which can excellently delete the HIV provirus from the infected human T cell genome. First, we designed zinc-finger nucleases (ZFNs) that target a sequence within the long terminal repeat (LTR) U3 region that is highly conserved in whole clade. Then, we screened out one pair of ZFN and named it as ZFN-U3. We discovered that ZFN-U3 can exactly target and eliminate the full-length HIV-1 proviral DNA after the infected human cell lines treated with it, and the frequency of its excision was about 30 % without cytotoxicity. These results prove that ZFN-U3 can efficiently excise integrated HIV-1 from the human genome in infected cells. This method to delete full length HIV-1 in human genome can therefore provide a novel approach to cure HIV-infected individuals in the future.
BOREAS TF-10 NSA-Fen Tower Flux and Meteorological Data

Science.gov (United States)

McCaughey, J. Harry; Hall, Forrest G. (Editor); Huemmrich, Karl (Editor); Jelinski, Dennis E.

2000-01-01

The BOREAS TF-10 team collected tower flux and meteorological data at two sites, a fen and a young jack pine forest, near Thompson, Manitoba, Canada, as part of BOREAS. A preliminary data set was assembled in August 1993 while field testing the instrument packages, and at both sites data were collected from 15-Aug to 31-Aug. The main experimental period was in 1994, when continuous data were collected from 08-Apr to 23-Sep at the fen site. A very limited experiment was run in the spring/ summer of 1995, when the fen site tower was operated from 08-Apr to 14-Jun in support of a hydrology experiment in an adjoining feeder basin. Upon examination of the 1994 data set, it became clear that the behavior of the heat, water, and carbon dioxide fluxes throughout the whole growing season was an important scientific question, and that the 1994 data record was not sufficiently long to capture the character of the seasonal behavior of the fluxes. Thus, the fen site was operated in 1996 in order to collect data from spring melt to autumn freeze-up. Data were collected from 29-Apr to 05-Nov at the fen site. All variables are presented as 30-minute averages. The data are stored in tabular ASCII files.
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.
The response of soil organic carbon of a rich fen peatland in interior Alaska to projected climate change.

Science.gov (United States)

Fan, Zhaosheng; David McGuire, Anthony; Turetsky, Merritt R; Harden, Jennifer W; Michael Waddington, James; Kane, Evan S

2013-02-01

It is important to understand the fate of carbon in boreal peatland soils in response to climate change because a substantial change in release of this carbon as CO2 and CH4 could influence the climate system. The goal of this research was to synthesize the results of a field water table manipulation experiment conducted in a boreal rich fen into a process-based model to understand how soil organic carbon (SOC) of the rich fen might respond to projected climate change. This model, the peatland version of the dynamic organic soil Terrestrial Ecosystem Model (peatland DOS-TEM), was calibrated with data collected during 2005-2011 from the control treatment of a boreal rich fen in the Alaska Peatland Experiment (APEX). The performance of the model was validated with the experimental data measured from the raised and lowered water-table treatments of APEX during the same period. The model was then applied to simulate future SOC dynamics of the rich fen control site under various CO2 emission scenarios. The results across these emissions scenarios suggest that the rate of SOC sequestration in the rich fen will increase between year 2012 and 2061 because the effects of warming increase heterotrophic respiration less than they increase carbon inputs via production. However, after 2061, the rate of SOC sequestration will be weakened and, as a result, the rich fen will likely become a carbon source to the atmosphere between 2062 and 2099. During this period, the effects of projected warming increase respiration so that it is greater than carbon inputs via production. Although changes in precipitation alone had relatively little effect on the dynamics of SOC, changes in precipitation did interact with warming to influence SOC dynamics for some climate scenarios. © 2012 Blackwell Publishing Ltd.
The response of soil organic carbon of a rich fen peatland in interior Alaska to projected climate change

Science.gov (United States)

Fan, Zhaosheng; McGuire, Anthony David; Turetsky, Merritt R.; Harden, Jennifer W.; Waddington, James Michael; Kane, Evan S.

2013-01-01

It is important to understand the fate of carbon in boreal peatland soils in response to climate change because a substantial change in release of this carbon as CO2 and CH4 could influence the climate system. The goal of this research was to synthesize the results of a field water table manipulation experiment conducted in a boreal rich fen into a process-based model to understand how soil organic carbon (SOC) of the rich fen might respond to projected climate change. This model, the peatland version of the dynamic organic soil Terrestrial Ecosystem Model (peatland DOS-TEM), was calibrated with data collected during 2005–2011 from the control treatment of a boreal rich fen in the Alaska Peatland Experiment (APEX). The performance of the model was validated with the experimental data measured from the raised and lowered water-table treatments of APEX during the same period. The model was then applied to simulate future SOC dynamics of the rich fen control site under various CO2 emission scenarios. The results across these emissions scenarios suggest that the rate of SOC sequestration in the rich fen will increase between year 2012 and 2061 because the effects of warming increase heterotrophic respiration less than they increase carbon inputs via production. However, after 2061, the rate of SOC sequestration will be weakened and, as a result, the rich fen will likely become a carbon source to the atmosphere between 2062 and 2099. During this period, the effects of projected warming increase respiration so that it is greater than carbon inputs via production. Although changes in precipitation alone had relatively little effect on the dynamics of SOC, changes in precipitation did interact with warming to influence SOC dynamics for some climate scenarios.
Fenómeno de Raynaud. Revisión

OpenAIRE

Jairo Alonso Rodríguez-Criollo; Daniel Jaramillo-Arroyave

2014-01-01

El fenómeno de Raynaud es una respuesta vascular exagerada al frio o al estrés que se manifiesta por cambios de coloración de la piel en diversas localizaciones, característicamente mostrando tres fases distintas que comprenden palidez, cianosis y rubor. La fisiopatología tiene varios componentes y puede tener varias etiologías, que comprenden vasoconstricción, agregación plaquetaria, trombosis y proliferación vascular. Dependiendo de la ausencia o la presencia de enfermedad de base, el fenóm...
Understanding toxicity at the watershed scale : design of the Syncrude Sandhill Fen watershed research project

International Nuclear Information System (INIS)

Wytrykush, C.

2010-01-01

Fens are peat-accumulating wetlands with a water table consisting of mineral-rich ground or surface water. This study discussed the construction of a fen-type reclaimed wetland constructed in a post-mining oil sands landscape. Syncrude Canada's Sandhill fen watershed project represents the first attempt at constructing a fen wetland in the oil sands region. The wetland and its watershed will be constructed on a soft tailings deposit. The design basis for the fen and watershed was developed by a team of researchers and scientists. The aim of the fen design was to control the salinity caused by tailings consolidation and seepage over time. Methods of mitigating potentially toxic effects from salinity were discussed.
The effect of an exceptionally wet summer on methane effluxes from a 15-year re-wetted fen in north-east Germany

Directory of Open Access Journals (Sweden)

V. Huth

2013-10-01

Full Text Available Re-wetting minerotrophic fens has become an important strategy to mitigate climate change in Germany. However, recent studies report raised methane (CH4 effluxes during the first years after flooding. A minerotrophic fen in north-east Germany that was re-wetted 15 years ago was exposed to exceptionally heavy rainfall and freshwater flooding in August 2011. We measured CH4 effluxes from wetland vegetation stands dominated by Phragmites australis (Cav. Trin. ex Steud., Typha latifolia L. and Carex acutiformis Ehrh., using the closed-chamber method, fortnightly from March 2011 to March 2012 with extra sampling during the flooding. The respective annual effluxes of CH4 (mean ± 1 standard error from the three vegetation types were 18.5 ± 1.3, 21.1 ± 1.2 and 47.5 ± 5.0 g m-2 a-1, with the August effluxes contributing 40 %, 50 % and 10 % of the annual effluxes. Despite the freshwater flooding in August, annual CH4 effluxes from the 15-year re-wetted fen are similar to those reported from pristine fens. These results are promising because they indicate that, although CH4 effluxes are elevated after re-wetting, they may return to values typical for pristine fens after 15 years. Hence, re-wetting can achieve the purpose of reducing greenhouse gas effluxes from drained minerotrophic fens.
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
HETEROGENEITY OF POLYCLONAL IMMUNOGLOBULINS NUCLEASE ACTIVITY IN RHEUMATOID AND REACTIVE ARTHRITIS

Directory of Open Access Journals (Sweden)

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
El fenómeno de bullying en Colombia

Directory of Open Access Journals (Sweden)

Ángela Paola Contreras Álvarez

2013-01-01

Full Text Available El presente artículo describe el nefasto fenómeno de violencia y hostigamiento en ambientes escolares denominado bullying o matoneo escolar, el cual, aunque ha sido analizado desde finales de los años setenta en los países escandinavos, tuvo auge en Colombia en el año 2000, adquiriendo fuerza y permanencia con el uso de redes sociales, evidenciando la falta de políticas nacionales educativas para la atención e intervención del problema, al igual que el olvido de educación en derechos humanos y resolución pacífica de conflictos en los colegios. Esta problemática se desarrolla a través del significado del bullying, la descripción de sus antecedentes, descripción general del fenómeno, relación de los deberes del Estado frente al tema, la concepción del fenómeno desde la perspectiva de la Corte Constitucional, concluyendo en algunas sugerencias para abordar la problemática escolar.
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.
TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells

Science.gov (United States)

Pedersen, Rune Troelsgaard; Kruse, Thomas; Nilsson, Jakob

2015-01-01

Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis. PMID:26283799
HYDRO-ECOLOGICAL ANALYSIS OF THE FEN SYSTEM LIEPER POSSE, EASTERN GERMANY

NARCIS (Netherlands)

VANDIGGELEN, R; GROOTJANS, AP; KEMMERS, RH; KOOIJMAN, AM; SUCCOW, M; DEVRIES, NPJ; VANWIRDUM, G; Vries, N.P.J. de; Wirdum, G. van

The Lieper Posse is a calcareous, ground water-fed fen system, situated in the end-moraine area of northeastern Germany. Nowadays the fen is partly drained and the vegetation adversely affected; hence regeneration measures are considered. Here we report a pilot survey of the actual vegetation cover
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...
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
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...
The hydrological and geochemical isolation of a freshwater bog within a saline fen in north-eastern Alberta

Directory of Open Access Journals (Sweden)

S.J. Scarlett

2013-10-01

Full Text Available In the oil sands development region near Fort McMurray, Alberta, wetlands cover ~62 % of the landscape, and ~95 % of these wetlands are peatlands. A saline fen was studied as a reference site for peatland reclamation. Despite highly saline conditions, a freshwater bog was observed in the path of local saline groundwater flow. The purpose of this study was to identify the hydrological controls that have allowed the development and persistence of a bog in this setting. The presence of bog vegetation and its dilute water chemistry suggest that saline groundwater from the fen rarely enters the bog, which functions predominantly as a groundwater recharge system. Chloride (Cl– and sodium (Na+ were the dominant ions in fen water, with concentrations averaging 5394 and 2307 mg L-1, respectively, while the concentrations in bog water were 5 and 4 mg L-1, respectively. These concentrations were reflected by salinity and electrical conductivity measurements, which in the fen averaged 9.3 ppt, and 15.8 mS cm-1, respectively, and in the bog averaged 0.1 ppt and 0.3 mS cm-1, respectively. A small ridge in the mineral substratum was found at the fen–bog margin, which created a persistent groundwater mound. Under the dry conditions experienced in early summer, groundwater flow was directed away from the bog at a rate of 14.6 mm day-1. The convex water table at the fen-bog margin impeded flow of saline water into the bog and instead directed it around the bog margin. However, the groundwater mound was eliminated during flooding in autumn, when the horizontal hydraulic gradient across the margin became negligible, suggesting the possibility of saline water ingress into the bog under these conditions.
Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease.

Directory of Open Access Journals (Sweden)

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.
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

Conservation and Biodiversity of Rich Fens

DEFF Research Database (Denmark)

Andersen, Dagmar Kappel

2014-01-01

Rich fen is a habitat type dependent on a constant supply of nutrient poor, calcium rich groundwater. A high, stable groundwater table, relatively high pH combined with nutrient poor conditions support a special and very species rich vegetation including many rare and threatened plant species. In...
A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

Energy Technology Data Exchange (ETDEWEB)

Fichorova, Raina N., E-mail: rfichorova@rics.bwh.harvard.edu [Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Mendonca, Kevin; Yamamoto, Hidemi S.; Murray, Ryan [Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Chandra, Neelima; Doncel, Gustavo F. [CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA (United States)

2015-06-15

Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV + 2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P < 0.05), and decreased levels of TLR2 (P < 0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P < 0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product. - Highlights: • A transcriptome nuclease protection assay assessed microbicides for vaginal safety. • Biomarkers were
A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

International Nuclear Information System (INIS)

Fichorova, Raina N.; Mendonca, Kevin; Yamamoto, Hidemi S.; Murray, Ryan; Chandra, Neelima; Doncel, Gustavo F.

2015-01-01

Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV + 2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P < 0.05), and decreased levels of TLR2 (P < 0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P < 0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product. - Highlights: • A transcriptome nuclease protection assay assessed microbicides for vaginal safety. • Biomarkers were
Examining the peat-accumulating potential of fen vegetation in the context of fed restoration of harvested peatlands

Energy Technology Data Exchange (ETDEWEB)

Graf, M.; Rochefort, L. [Laval Univ., Quebec City, PQ (Canada). Dept. de Phytologie, Peatland Ecology and Research Group

2009-07-01

The return of peat-accumulating function is an important long-term goal in peatland restoration. However, researchers do not yet know which vegetation groups increase the ability of ecosystems to accumulate peat in fens. This study assessed the decomposition rates of 3 vegetation species typical to undisturbed fen regions, and 3 species that spontaneously colonize harvested fens. All species were incubated in a harvest fen restoration site as well as at an undisturbed fen in order to compare decomposition rates according to different environmental conditions. The study showed that the average exponential decay coefficient for all species was higher at the harvested fen site. The decay coefficient varied only slightly between undisturbed and harvested fens, despite wide variations in hydrological conditions. Litter type had the most significant impact on decomposition rates. Annual primary production of the species was also measured in order to estimate the peat accumulating capacity of each species. Results showed that Scirpus cyperinus primary annual production rates were 3 times higher than the other studied species. It was concluded that the harvested fen sites have a higher peat-accumulating potential than undisturbed fens. 60 refs., 2 tabs., 2 figs.
Design, construction and performance of an experimental watershed to support a fen peatland for mine landscape reclamation

Science.gov (United States)

Price, J. S.; Petrone, R. M.; Strack, M.; Cooper, D. J.

2017-12-01

In the Alberta oil sands region, fen peatlands comprised 50% of the boreal landscape. Oil sands mining has stripped over 800 km2 of land surface to access bitumen, necessitating landscape reclamation to re-establish functional wetlands. Fens are peat-dominated wetlands that commonly rely on groundwater to supplement their water budget and deliver dissolved solutes that impart a distinct geochemistry, hence vegetation community. A numerical model was used to test the concept and guide selection of earth materials and system geometry. The goal was to maintain the placed peat in a sufficiently wet condition to support wetland plants and become a net carbon sink, in this sub-humid climate. The 32.1 ha Nikanotee Fen Watershed comprises a 7.7 ha upland, that was designed to recharge sufficient water, and deliver it to the 2.9 ha fen via groundwater flow. These features are surrounded by other reclaimed slopes designed to store water, rather than deliver it downslope. Four years of monitoring since construction show the fen maintains a high water table, and the peatland has become a strong carbon sink, even though the hydrological performance of construction materials varied substantially from what was anticipated (lower hydraulic conductivity). However, solutes associated with the tailings sand used in construction are moving towards the fen, and are expected to influence the future vegetation community and system biogeochemistry. One of the biggest uncertainties is the changing performance of soils and vegetation as they develop.
Decomposition of Carex and Sphagnum litter in two mesotrophic fens differing in dominant plant species

NARCIS (Netherlands)

Scheffer, R.A.; Van Logtestijn, R. S P; Verhoeven, J. T A

2001-01-01

Peatlands can be classified into fens and bogs based on their hydrology. Development of fens to bogs is accompanied by the invasion of Sphagnum species. The purpose of this study was to determine how the decomposition process in fens is influenced by the transition from a vascular plant-dominated
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
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.
Compostos fenólicos totais e taninos condensados em frutas nativas do cerrado

Directory of Open Access Journals (Sweden)

Wesley Silveira Rocha

2011-12-01

Full Text Available As frutas nativas do cerrado têm despertado ultimamente interesse crescente, devido às suas propriedades nutricionais e funcionais aliadas ao potencial para agregar valor e conservar a biodiversidade deste bioma. Muitos compostos fenólicos apresentam capacidade antioxidante de neutralizar a atividade de radicais livres gerados no organismo, que estão associados a diversas doenças crônico-degenerativas. O objetivo deste trabalho foi avaliar a eficiência de três soluções extratoras (acetona a 70 %, etanol a 95 % e metanol a 99,8 % e determinar os teores de compostos fenólicos em 10 espécies de frutas nativas do cerrado, utilizando o método de Folin-Ciocalteou para compostos fenólicos totais e o método da vanilina para taninos condensados. A acetona a 70 % mostrou-se como o melhor solvente extrator de compostos fenólicos totais e taninos condensados em frutos de Pouteria gardneriana, Eugenia dysenterica, E. klostzchiana, E. punicifolia, Plinia edulis, Campomanesia sp., fenólicos totais de Brosimium gaudichaudii e taninos condensados de Jaracatia spinosa. O etanol a 95 % foi mais eficiente na extração de fenólicos totais de Jaracatia spinosa e taninos condensados de Brosimium gaudichaudii; o metanol a 99,8 % foi mais eficiente na extração de taninos condensados de Eugenia dysenterica. Os teores de compostos fenólicos totais variaram entre 90 e 327 mg de ácido gálico equivalente por 100g de polpa para as espécies E. dysenterica e E. punicifolia, respectivamente. Os teores de taninos condensados variaram entre 4 e 291 mg de catequina equivalente por 100 g de polpa para as espécies E. dysenterica e E. calcyna, respectivamente. As espécies de frutas do cerrado, avaliadas neste estudo, podem ser consideradas boas fontes de compostos fenólicos totais, sendo que a natureza específica dos diferentes tipos deve ser avaliada em estudos futuros.
Determination of natural organic matter and iron binding capacity in fen samples

Science.gov (United States)

Kügler, Stefan; Cooper, Rebecca E.; Frieder Mohr, Jan; Wichard, Thomas; Küsel, Kirsten

2017-04-01

Natural organic matter (NOM) plays an important role in ecosystem processes such as soil carbon stabilization, nutrient availability and metal complexation. Iron-NOM-complexes, for example, are known to increase the solubility and, as a result, the bioavailability of iron in natural environments leading to several effects on the microbial community. Due to the various functions of NOM in natural environments, there is a high level of interest in the characterization of the molecular composition. The complexity of NOM presents a significant challenge in the elucidation of its composition. However, the development and utilization of high resolution mass spectrometry (HR-MS) as a tool to detect single compounds in complex samples has spearheaded the effort to elucidate the composition of NOM. Over the past years, the accuracy of ion cyclotron- or Orbitrap mass spectrometers has increased dramatically resulting in the possibility to obtain a mass differentiation of the large number of compounds in NOM. Together these tools provide significant and powerful insight into the molecular composition of NOM. In the current study, we aim to understand abiotic and biotic interactions between NOM and metals, such as iron, found in the Schlöppnerbrunnen fen (Fichtelgebirge, Germany) and how these interactions impact the microbial consortia. We characterized the dissolved organic matter (DOM) as well as basic chemical parameters in the iron-rich (up to 20 mg (g wt peat)-1), slightly acidic (pH 4.8) fen to gain more information about DOM-metal interactions. This minerotrophic peatland connected to the groundwater has received Fe(II) released from the surrounding soils in the Lehstenbach catchment. Absorption spectroscopy (AAS), differential pulse polarography (DPP) and high resolution electrospray ionization mass spectrometry (HR-ESI-Orbitrap-MS) was applied to characterize the molecular composition of DOM in the peat water extract (PWE). We identified typical patterns for DOM
compuestos fenólicos

Directory of Open Access Journals (Sweden)

Yolanda González García

2005-01-01

Full Text Available En este trabajo se presenta una revisión bibliográfica que tiene como objetivo identificar y analizar alternativas de aprovechamiento del bagazo de Agave tequilana Weber var. Azul. Se proponen dos líneas de investigación para obtener productos de valor agregado a partir de este residuo: 1utilización como fuente de carbono de bajo costo para la producción de biopolímeros y carbohidrasas, con bacterias celulolíticas del género Microbulbifer, Marinobacterium y Sagittula; 2obtención de compuestos fenólicos a partir de la lignina presente en sus fibras. Se concluye, que el bagazo de agave tequilero es un material viable para ser usado como substrato en procesos fermentativos, por lo que existen grandes oportunidades de investigación para desarrollar procesos de bioconversión técnicamente factible, eficiente y económicamente atractivo
Water level, vegetation composition, and plant productivity explain greenhouse gas fluxes in temperate cutover fens after inundation

Science.gov (United States)

Minke, Merten; Augustin, Jürgen; Burlo, Andrei; Yarmashuk, Tatsiana; Chuvashova, Hanna; Thiele, Annett; Freibauer, Annette; Tikhonov, Vitalij; Hoffmann, Mathias

2016-07-01

Peat extraction leaves a land surface with a strong relief of deep cutover areas and higher ridges. Rewetting inundates the deep parts, while less deeply extracted zones remain at or above the water level. In temperate fens the flooded areas are colonized by helophytes such as Eriophorum angustifolium, Carex spp., Typha latifolia or Phragmites australis dependent on water depth. Reeds of Typha and Phragmites are reported as large sources of methane, but data on net CO2 uptake are contradictory for Typha and rare for Phragmites. Here, we analyze the effect of vegetation, water level and nutrient conditions on greenhouse gas (GHG) emissions for representative vegetation types along water level gradients at two rewetted cutover fens (mesotrophic and eutrophic) in Belarus. Greenhouse gas emissions were measured campaign-wise with manual chambers every 2 to 4 weeks for 2 years and interpolated by modelling. All sites had negligible nitrous oxide exchange rates. Most sites were carbon sinks and small GHG sources. Methane emissions generally increased with net ecosystem CO2 uptake. Mesotrophic small sedge reeds with water table around the land surface were small GHG sources in the range of 2.3 to 4.2 t CO2 eq. ha-1 yr-1. Eutrophic tall sedge - Typha latifolia reeds on newly formed floating mats were substantial net GHG emitters in the range of 25.1 to 39.1 t CO2 eq. ha-1 yr. They represent transient vegetation stages. Phragmites reeds ranged between -1.7 to 4.2 t CO2 eq. ha-1 yr-1 with an overall mean GHG emission of 1.3 t CO2 eq. ha-1 yr-1. The annual CO2 balance was best explained by vegetation biomass, which includes the role of vegetation composition and species. Methane emissions were obviously driven by biological activity of vegetation and soil organisms. Shallow flooding of cutover temperate fens is a suitable measure to arrive at low GHG emissions. Phragmites australis establishment should be promoted in deeper flooded areas and will lead to moderate, but
Seasonal Trace Gas Dynamics on Minerotrophic Fen Peatlands in NE-Germany

Science.gov (United States)

Giebels, Michael; Beyer, Madlen; Augustin, Jürgen; Minke, Merten; Juszczak, Radoszlav; Serba, Tomasz

2010-05-01

In Germany more than 99 % of fens have lost their carbon and nutrient sink function due to heavy drainage and agricultural land use especially during the last decades and thus resulted in compression and heavy peat loss (CHARMAN 2002; JOOSTEN & CLARKE 2002; SUCCOW & JOOSTEN 2001; AUGUSTIN et al. 1996; KUNTZE 1993). Therefore fen peatlands play an important part (4-5 %) in the national anthropogenic trace gas budget. But only a small part of drained and agricultural used fens in NE Germany can be restored. Knowledge of the influence of land use to trace gas exchange is important for mitigation of the climate impact of the anthropogenic peatland use. We study carbon exchanges of several fen peatland use areas between soil and atmosphere at different sites in NE-Germany. Our research covers peatlands of supposed strongly climate forcing land use (cornfield and intensive pasture) and of probably less forcing, alternative types (meadow and extensive pasture) as well as rewetted (formerly drained) areas and near-natural sites like a low-degraded fen and a wetted alder woodland. We measured trace gas fluxes with manual and automatic chambers in periodic routines since spring 2007. The used chamber technique bases on DROESLER (2005). In total we now do research at 22 sites situated in 5 different locations covering agricultural, varying states of rewetted and near-natural treatments. We present results of at least 2 years of measurements and show significant differences in their annual trace gas balances depending on the genesis of the observed sites and the seasonal dynamics. Crosswise comparison of different site treatments combined with the seasonal environmental observations give good hints for the identification of main flux driving parameters. That is that a reduced intensity in land use as a supposed mitigating treatment did not show the expected effect, though a normal meadow treatment surprisingly resulted in the lowest balances in both years. For implementing a
Seasonal Carbon Dynamics on Selected Fen Peatland Sites in NE-Germany

Science.gov (United States)

Giebels, Michael; Beyer, Madlen; Augustin, Jürgen; Minke, Merten; Juszczak, Radoszlav; Serba, Tomasz

2010-05-01

In Germany more than 99 % of fens have lost their carbon and nutrient sink function due to heavy drainage and agricultural land use especially during the last decades and thus resulted in compression and heavy peat loss (CHARMAN 2002; JOOSTEN & CLARKE 2002; SUCCOW & JOOSTEN 2001; AUGUSTIN et al. 1996; KUNTZE 1993). Therefore fen peatlands play an important part (4-5 %) in the national anthropogenic trace gas budget. But only a small part of drained and agricultural used fens in NE Germany can be restored. Knowledge of the influence of land use to trace gas exchange is important for mitigation of the climate impact of the anthropogenic peatland use. We study carbon exchanges of several fen peatland use areas between soil and atmosphere at different sites in NE-Germany. Our research covers peatlands of supposed strongly climate forcing land use (cornfield and intensive pasture) and of probably less forcing, alternative types (meadow and extensive pasture) as well as rewetted (formerly drained) areas and near-natural sites like a low-degraded fen and a wetted alder woodland. We measured trace gas fluxes with manual and automatic chambers in periodic routines since spring 2007. The used chamber technique bases on DROESLER (2005). In total we now do research at 22 sites situated in 5 different locations covering agricultural, varying states of rewetted and near-natural treatments. We present results of at least 2 years of measurements and show significant differences in their annual carbon balances depending on the genesis of the observed sites and the seasonal dynamics. Crosswise comparison of different site treatments combined with the seasonal environmental observations give good hints for the identification of main flux driving parameters. That is that a reduced intensity in land use as a supposed mitigating treatment did not show the expected effect, though a normal meadow treatment surprisingly resulted in the lowest CO2 balances in both years. For implementing a
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...
Complex microbiota of a Chinese "Fen" liquor fermentation starter (Fen-Daqu), revealed by culture-dependent and culture-independent methods

NARCIS (Netherlands)

Zheng, X.; Zheng, Y.; Han, B.; Zwietering, M.H.; Samson, R.A.; Boekhout, T.; Nout, M.J.R.

2012-01-01

Daqu is a traditional fermentation starter that is used for Chinese liquor production. Although partly mechanized, its manufacturing process has remained traditional. We investigated the microbial diversity of Fen-Daqu, a starter for light-flavour liquor, using combined culture-dependent and
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
Distribution of ultraviolet-induced DNA repair synthesis in nuclease sensitive and resistant regions of human chromatin

International Nuclear Information System (INIS)

Smerdon, M.J.; Tlsty, T.D.; Lieberman, M.W.

1978-01-01

The distribution of ultraviolet radiation (uv) induced DNA repair synthesis within chromatin was examined in cultured human diploid fibroblasts (IMR-90). Measurement of the time course of repair synthesis yielded two distinct phases: An initial rapid phase (fast repair) which occurs during the first 2 to 3 h after damage and a slower phase (slow repair) associated with a tenfold decrease in the rate of nucleotide incorporation, which persists for at least 35 h after damage. Staphylococcal nuclease digests of nuclei from cells damaged with uv and labeled during the fast-repair phase revealed a marked preference of fast-repair synthesis for the nuclease-sensitive regions. A new method was developed to analyze the digestion data and showed that approximately 50% of the nucleotides incorporated during the fast-repair phase are located in staphylococcal nuclease-sensitive regions, which comprise about 30% of the genome. Calculations from these data indicate that in the staphylococcal nuclease-sensitive regions the number of newly inserted nucleotides per unit DNA is about twice that of resistant regions. These results were supported by electrophoresis studies which demonstrated a decreased representation of fast-repair synthesis in core particle DNA. In contrast, the distribution within chromatin of nucleotides incorporated during the slow-repair phase was found to be much more homogeneous with about 30% of the repair sites located in 25% of the genome. Digestion studieswith DNase I indicated a slight preference of repair synthesis for regions sensitive to this enzyme; however, no marked difference between the distributions of fast- and slow-repair synthesis was observed. This study provides evidence that the structural constraints placed upon DNA in chromatin also place constraints upon uv-induced DNA repair synthesis in human cells
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

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
Filtering fens: mechanisms explaining phosphorus-limited hotspots of biodiversity in wetlands adjacent to heavily fertilized areas.

Science.gov (United States)

Cusell, Casper; Kooijman, Annemieke; Fernandez, Filippo; van Wirdum, Geert; Geurts, Jeroen J M; van Loon, E Emiel; Kalbitz, Karsten; Lamers, Leon P M

2014-05-15

The conservation of biodiverse wetland vegetation, including that of rich fens, has a high priority at a global scale. Although P-eutrophication may strongly decrease biodiversity in rich fens, some well-developed habitats do still survive in highly fertilized regions due to nutrient filtering services of large wetlands. The occurrence of such nutrient gradients is well-known, but the biogeochemical mechanisms that determine these patterns are often unclear. We therefore analyzed chemical speciation and binding of relevant nutrients and minerals in surface waters, soils and plants along such gradients in the large Ramsar nature reserve Weerribben-Wieden in the Netherlands. P-availability was lowest in relatively isolated floating rich fens, where plant N:P ratios indicated P-limitation. P-limitation can persist here despite high P-concentrations in surface waters near the peripheral entry locations, because only a small part of the P-input reaches the more isolated waters and fens. This pattern in P-availability appears to be primarily due to precipitation of Fe-phosphates, which mainly occurs close to entry locations as indicated by decreasing concentrations of Fe- and Al-bound P in the sub-aquatic sediments along this gradient. A further decrease of P-availability is caused by biological sequestration, which occurs throughout the wetland as indicated by equal concentrations of organic P in all sub-aquatic sediments. Our results clearly show that the periphery of large wetlands does indeed act as an efficient P-filter, sustaining the necessary P-limitation in more isolated parts. However, this filtering function does harm the ecological quality of the peripheral parts of the reserve. The filtering mechanisms, such as precipitation of Fe-phosphates and biological uptake of P, are crucial for the conservation and restoration of biodiverse rich fens in wetlands that receive eutrophic water from their surroundings. This seems to implicate that biodiverse wetland
Remotely sensed hydrological isolation : a key factor predicting plant species distribution in fens

NARCIS (Netherlands)

Sanders, M.E.

1999-01-01

In fens the species composition, vegetation structure and succession rate are determined by vegetation management and water chemistry, particularly by the base status and nutrient status. Base-rich and nutrient-rich surface water causes fens to become eutrophied, which leads to an increased
Investigation of centers sensitive to S1-nuclease in the genoma of the yeast S. cerevisiae after in-vivo exposure to gamma radiation

International Nuclear Information System (INIS)

Geigl, E.M.

1987-09-01

The structure, distribution and repair of basal damage in DNS after exposure to 60 Co gamma radiation were investigated in S. cerevisiae cells. Small DNS regions with mispaired or unpaired bases of rather high stability were found whose rate of incidence and linear dose dependence appear to be similar to those of double strand breaks. In contrast to double strand breaks, they showed no statistical' distribution pattern across the genoma. Liquid holding experiments showed that centers sensitive to S1-nuclease will be repaired in S. cerevisiae by a combined process of recombination and postreplication repair; the gene products of the genes RAD50 and RAD18 are involved. (orig./AJ) [de
Modelado lingüístico de fenómenos complejos

OpenAIRE

Álvarez Álvarez, Alberto

2012-01-01

Actualmente, es posible adquirir y almacenar grandes volúmenes de datos sobre diferentes fenómenos complejos en muchas áreas importantes. Con el fin de ser útil, esta información debe ser explicada de la manera más comprensible posible, incluyendo los conocimientos previos disponibles sobre el fenómeno que se estudia. Estos objetivos sólo pueden lograrse mediante el uso de lenguaje natural, especialmente si la información final va a ser utilizada por personas no expertas. Por lo tanto, e...
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.
Diferentes Abordagens dos Fenômenos Subjetivos na Obra de B. F. Skinner

Directory of Open Access Journals (Sweden)

Henrique Mesquita Pompermaier

2014-08-01

Full Text Available Como a análise do comportamento aborda os fenômenos subjetivos? Seria a teoria de eventos privados a única maneira? Guiado por essas questões, o presente trabalho busca apresentar a abordagem dos fenômenos subjetivos na obra de B. F. Skinner, indicando não apenas a teoria de eventos privados, mas também a possibilidade de abordagens alternativas a essa temática, sem referência à privacidade, por exemplo, por meio da magnitude das ações envolvidas nos fenômenos, níveis (aberto e encoberto do comportamento e conceitos disposicionais. Indica-se ainda uma significativa diferença de ênfase na teoria de eventos privados ao longo da obra skinneriana. Por fim, conduz-se uma discussão sobre o papel da teoria de eventos privados na análise do comportamento para a abordagem dos fenômenos subjetivos.
NanoRNase from Aeropyrum pernix shows nuclease activity on ssDNA and ssRNA.

Science.gov (United States)

Deng, Yong-Jie; Feng, Lei; Zhou, Huan; Xiao, Xiang; Wang, Feng-Ping; Liu, Xi-Peng

2018-05-01

In cells, degrading DNA and RNA by various nucleases is very important. These processes are strictly controlled and regulated to maintain DNA integrity and to mature or recycle various RNAs. NanoRNase (Nrn) is a 3'-exonuclease that specifically degrades nanoRNAs shorter than 5 nucleotides. Several Nrns have been identified and characterized in bacteria, mainly in Firmicutes. Archaea often grow in extreme environments and might be subjected to more damage to DNA/RNA, so DNA repair and recycling of damaged RNA are very important in archaea. There is no report on the identification and characterization of Nrn in archaea. Aeropyrum pernix encodes three potential Nrns: NrnA (Ape1437), NrnB (Ape0124), and an Nrn-like protein Ape2190. Biochemical characterization showed that only Ape0124 could degrade ssDNA and ssRNA from the 3'-end in the presence of Mn 2+ . Interestingly, unlike bacterial Nrns, Ape0124 prefers ssDNA, including short nanoDNA, and degrades nanoRNA with lower efficiency. The 3'-DNA backbone was found to be required for efficiently hydrolyzing the phosphodiester bonds. In addition, Ape0124 also degrads the 3'-overhang of double-stranded DNA. Interestingly, Ape0124 could hydrolyze pAp into AMP, which is a feature of bacterial NrnA, not NrnB. Our results indicate that Ape0124 is a novel Nrn with a combined substrate profile of bacterial NrnA and NrnB. Copyright © 2018 Elsevier B.V. All rights reserved.
El Fenómeno de Consumo y el Consumo en Marketing

Directory of Open Access Journals (Sweden)

Dagoberto Páramo Morales

2004-01-01

Full Text Available En este artículo se presentan las principales características que tipifican el fenómeno de consumo en las sociedades contemporáneas. Para lograrlo, primero se revisan algunas aproximaciones conceptuales del consumo, visto como fenómeno social, asociándolo con el simbolismo de consumo, los rituales, las posesiones y el materialismo como motores propulsores de la concepción y práctica del marketing de hoy.
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...
Fenólicos totais e capacidade antioxidante in vitro de polpas de frutos tropicais

Directory of Open Access Journals (Sweden)

Luanne Morais Vieira

2011-09-01

Full Text Available O consumo de frutos e suas polpas tem sido muito recomendado por seu valor nutricional, alto teor de fibras, vitamina C e carotenoides. Trabalhos recentes têm apontado esses alimentos como fontes de compostos fenólicos com ação antioxidante, portanto sequestradores de radicais livres, com ação protetora contra o surgimento e/ou desenvolvimento de processos degenerativos que conduzem a doenças crônicas não transmissíveis. Devido à crescente comercialização e consumo de polpas de frutas no Brasil, especialmente na cidade de Teresina-Piauí, este trabalho selecionou um grupo de polpas de frutos de elevado consumo local para avaliação do teor de fenólicos totais e da atividade antioxidante in vitro pelo método de captura de radicais livres: DPPH (radical 1,1-diphenil-2-picrilhydrazil e ABTS (radical 2,2'azinobis(3-ethylbenzthiazoline-6-sulfonic acid. Os frutos selecionados foram: Acerola (Malpighia emarginata DC., Bacuri (Platonia insignis Mart., Cajá (Spondias mombin L., Caju (Anacardium occidentale, Goiaba(Psidium guajava e Tamarindo (Tamarindus indica L.. Os teores de fenólicos totais encontrados nas polpascongeladas destes frutos exibiram quantidades relevantes de polifenóis, destacando-se a polpa de acerola com 835,25 ± 32,44 e 449,63 ± 10,24 mg /100g nos extratos aquosos e hidroalcoólicos, respectivamente, seguido pela polpa de caju com 201,61 ± 19,15 e 165,07 ± 4,10 mg /100g. As polpas de bacuri e tamarindo foram as que apresentaram os menores teores de fenólicos totais. Com relação à atividade antioxidante in vitro, os melhores resultados foram encontrados para os extratos aquosos e hidroalcoólicos das polpas de acerola, caju e goiaba. A capacidade antioxidante destas polpas (EC50 em µg/mL variou de 24,42 a 413,36 e de 1,74 a 259,18 para os extratos aquosos e hidroalcoólicos, respectivamente. Utilizando o radical ABTS, a atividade antioxidante para essas mesmas polpas de frutas apresentou valores TEAC que
The annual carbon budget for fen and forest in a wetland at Arctic treeline

Energy Technology Data Exchange (ETDEWEB)

Rouse, W. R. [McMaster Univ., School of Geography and Geology, Hamilton, ON (Canada); Bello, R. L.; D' Souza, A. [York Univ., Dept. of Geography, Toronto, ON (Canada); Griffis, T. J. [Minnesota Univ., Dept. of Soil, Water and Climate, St. Paul, MN (United States); LaFleur, P. M. [Trent Univ., Dept. of Geography, Peterborough, ON (Canada)

2002-09-01

This study of a wetland system at the Arctic treeline compares two carbon budget estimates, one derived from long-term growth rates of organic soil and the other based on shorter-term flux measurements. Result showed that while there was a small loss of carbon from the ecosystem in the case of tundra fen over a period of fifty-three years, the adjacent open subarctic forest showed large gains in atmospheric carbon dioxide during the same period. These longer-term data were supported by shorter-term flux measurements, which also showed carbon loss by the fen and carbon uptake by the forest. The shorter term data indicate that the carbon loss from the fen during this period was attributable to one particularly dry year. The different rates of carbon exchange appear to be controlled by two primary factors. The first is the enhanced development in the fen of a photosynthesis-inhibiting hummock-hollow landscape, while the second is related to warmer and drier average growing season conditions, which inhibit carbon dioxide uptake in the fen and enhances it in the forest. 24 refs., 4 tabs., 3 figs.
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
Fenóis totais, flavonoides totais e atividade antioxidante de Selaginella convoluta (Arn. Spring (Selaginellaceae

Directory of Open Access Journals (Sweden)

Pedro Guilherme Sousa de Sá

2012-12-01

Full Text Available Selaginella convoluta é uma espécie conhecida no Nordeste do Brasil como “jericó”, e bastante utilizada na medicina popular para tratamento de doenças. Este estudo teve como objetivo determinar o teor de compostos fenólicos e avaliar a atividade antioxidante in vitro do extrato etanólico e das frações obtidas por partição de S. convoluta. O conteúdo de fenóis totais foi determinado pelo método de Folin-Ciocalteu. O teor de flavonoides totais também foi avaliado. A atividade antioxidante dos extratos foi avaliada usando o método do sequestro do radical DPPH e inibição da auto-oxidação do sistema β-caroteno-ácido linoleico e comparada com os compostos de referência ácido ascórbico, BHA, BHT, quercetina e pirogalol. O conteúdo fenólico total foi de 209,90 ± 19,84 e 61,13 ± 2,50 mg equivalente de ácido gálico/g para os extratos AcOEt e EEB, respectivamente. O conteúdo de flavonoides totais foi de 155,70 ± 6,21 e 62,13 ± 4,10 para os dois extratos, respectivamente. Os extratos AcOEt e EEB apresentaram boas atividades antioxidantes. BHA foi o antioxidante mais efetivo, com um valor de IC50 de 1,62 ± 0,69 µg/mL. Os resultados obtidos mostram que S. convoluta pode ser uma boa fonte de compostos fenólicos antioxidantes. Estudos posteriores serão realizados para se chegar ao isolamento e identificação dos principais constituintes fenólicos dos extratos.
The Cdc45/RecJ-like protein forms a complex with GINS and MCM, and is important for DNA replication in Thermococcus kodakarensis.

Science.gov (United States)

Nagata, Mariko; Ishino, Sonoko; Yamagami, Takeshi; Ogino, Hiromi; Simons, Jan-Robert; Kanai, Tamotsu; Atomi, Haruyuki; Ishino, Yoshizumi

2017-10-13

The archaeal minichromosome maintenance (MCM) has DNA helicase activity, which is stimulated by GINS in several archaea. In the eukaryotic replicative helicase complex, Cdc45 forms a complex with MCM and GINS, named as CMG (Cdc45-MCM-GINS). Cdc45 shares sequence similarity with bacterial RecJ. A Cdc45/RecJ-like protein from Thermococcus kodakarensis shows a bacterial RecJ-like exonuclease activity, which is stimulated by GINS in vitro. Therefore, this archaeal Cdc45/RecJ is designated as GAN, from GINS-associated nuclease. In this study, we identified the CMG-like complex in T. kodakarensis cells. The GAN·GINS complex stimulated the MCM helicase, but MCM did not affect the nuclease activity of GAN in vitro. The gene disruption analysis showed that GAN was non-essential for its viability but the Δgan mutant did not grow at 93°C. Furthermore, the Δgan mutant showed a clear retardation in growth as compared with the parent cells under optimal conditions at 85°C. These deficiencies were recovered by introducing the gan gene encoding the nuclease deficient GAN protein back to the genome. These results suggest that the replicative helicase complex without GAN may become unstable and ineffective in replication fork progression. The nuclease activity of GAN is not related to the growth defects of the Δgan mutant cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
Fenómenos de transporte : un curso introductorio

OpenAIRE

Betancourt Grajales, Ramiro

1991-01-01

Fenómenos de transporte, es el nombre colectivo que se da al estudio sistemático e integrado de tres áreas clásicas de la ciencia de la Ingeniería : 1) Transporte de Energía o Calor, 2) Transporte de Masa o Difusión, y 3) Transporte de Cantidad de Movimiento o Impulso (Momentum en Inglés), o Dinámica de Fluidos. Debido a que con frecuencia el transporte de masa y de calor ocurren en un fluido, algunos planes de estudio incluyen estos procesos en su tratamiento de la mecánica de fluidos....
Microbial activity and community structure in two drained fen soils in the Ljubljana Marsh

NARCIS (Netherlands)

Kraigher, Barbara; Stres, Blaz; Hacin, Janez; Ausec, Luka; Mahne, Ivan; van Elsas, Jan D.; Mandic-Mulec, Ines

Fen peatlands are specific wetland ecosystems containing high soil organic carbon (SOC). There is a general lack of knowledge about the microbial communities that abound in these systems. We examined the microbial activity and community structure in two fen soils differing in SOC content sampled
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.
Mechanisms controlling Cu, Fe, Mn, and Co profiles in peat of the Filson Creek Fen, northeastern Minnesota

Science.gov (United States)

Walton-Day, K.; Filipek, L.H.; Papp, C.S.E.

1990-01-01

Filson Creek Fen, located in northeastern Minnesota, overlies a Cu-Ni sulfide deposit. A site in the fen was studied to evaluate the hydrogeochemical mechanisms governing the development of Fe, Mn, Co, and Cu profiles in the peat. At the study site, surface peat approximately 1 m thick is separated from the underlying mineralized bedrock by a 6-12 m thickness of lake and glaciofluvial sediments and till. Concentrations of Fe, Mn, Co, and Cu in peat and major elements in pore water delineate a shallow, relatively oxidized, Cu-rich zone overlying a deeper, reduced, Fe-, Mn-, and Co-rich zone within the peat. Sequential metal extractions from peat samples reveal that 40-55% of the Cu in the shallow zone is associated with organic material, whereas the remaining Cu is distributed between iron-oxide, sulfide, and residual fractions. Sixty to seventy percent of the Fe, Mn, and Co concentrated in the deeper zone occur in the residual phase. The metal profiles and associations probably result from non-steady-state input of metals and detritus into the fen during formation of the peat column. The enrichment of organic-associated Cu in the upper, oxidized zone represents a combination of Cu transported into the fen with detrital plant fragments and soluble Cu, derived from weathering of outcrop and subcrop of the mineral deposit, transported into the fen, and fixed onto organic matter in the peat. The variable stratigraphy of the peat indicates that weathering processes and surface vegetation have changed through time in the fen. The Fe, Mn, and Co maxima at the base of the peat are associated with a maximum in detrital matter content of the peat resulting from a transition between the underlying inorganic sedimentary environment to an organic sedimentary environment. The chemistry of sediments and ground water collected beneath the peat indicate that mobilization of metals from sulfide minerals in the buried mineral deposit or glacial deposits is minimal. Therefore, the
Synergistic increase of oxygen reduction favourable Fe-N coordination structures in a ternary hybrid of carbon nanospheres/carbon nanotubes/graphene sheets.

Science.gov (United States)

Zhang, Shiming; Liu, Bin; Chen, Shengli

2013-11-14

A Fe/N co-doped ternary nanocarbon hybrid, with uniform bamboo-like carbon nanotubes (CNTs) in situ grown on/between the single/few-layer graphene sheets interspaced by carbon nanosphere aggregates, was prepared through a one-pot heat treatment of a precursor mixture containing graphene oxide, Vulcan XC-72 carbon nanospheres, nitrogen rich melamine and small amounts of Fe ions. Physical characterization including electron microscopic images, N2 adsorption-desorption isotherms, pore size distribution, XPS, XRD, Mössbauer spectra, and EDX revealed that the 0-D/1-D/2-D ternary hybrid architecture not only offered an optimized morphology for high dispersion of each nanocarbon moiety, while the carbon nanosphere interspaced graphene sheets have provided a platform for efficient reaction between Fe ions and melamine molecules, resulting in uniform nucleation and growth of CNTs and formation of high density Fe-N coordination assemblies that have been believed to be the active centers for the oxygen reduction reaction (ORR) in carbon-based nonprecious metal electrocatalysts. In the absence of graphene oxides or carbon nanospheres, a similar heat treatment was found to result in large amounts of elemental Fe and Fe carbides and entangled CNTs with wide diameter distributions. As a result, the ternary Fe/N-doped nanocarbon hybrid exhibits ORR activity much higher than the Fe-N doped single or binary nanocarbon materials prepared under similar heat treatment conditions, and approaching that of the state-of-the-art carbon-supported platinum catalyst (Pt/C) in acidic media, as well as superior stability and methanol tolerance to Pt/C.

From groundwater abstraction to vegetative response in fen ecosystems

DEFF Research Database (Denmark)

Johansen, Ole Munch; Jensen, Jacob Birk; Pedersen, Morten Lauge

2014-01-01

Hydrological effects of groundwater abstraction near a Danish river valley have been assessed by integrated hydrological modelling. The study site contains groundwater-dependent terrestrial ecosystems in terms of fen and spring habitats that are highly dependent on regional and local scale...... hydrology. Fens are rare and threatened worldwide due to pressures from agriculture, to lack of appropriate management and to altered catchment hydrology. A solid foundation for hydrological modelling was established based on intensive monitoring at the site, combined with full-scale pumping tests...... in the area. A regional groundwater model was used to describe the dynamics in groundwater recharge and the large-scale discharge to streams. A local grid refinement approach was then applied in a detailed assessment of damage in order to balance the computational effort and the need for a high spatial...
Water flow and solute transport in floating fen root mats

Science.gov (United States)

Stofberg, Sija F.; EATM van der Zee, Sjoerd

2015-04-01

be very similar and likely functionally related. Our experimental field data were used for modelling water flow and solute transport in floating fens, using HYDRUS 2D. Fluctuations of surface water and root mat, as well as geometry and unsaturated zone parameters can have a major influence on groundwater fluctuations and the exchange between rain and surface water and the water in the root mats. In combination with the duration of salt pulses in surface water, and sensitivity of fen plants to salinity (Stofberg et al. 2014, submitted), risks for rare plants can be anticipated.
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...
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.
Organization of the BcgI restriction-modification protein for the cleavage of eight phosphodiester bonds in DNA

Science.gov (United States)

Smith, Rachel M.; Marshall, Jacqueline J. T.; Jacklin, Alistair J.; Retter, Susan E.; Halford, Stephen E.; Sobott, Frank

2013-01-01

Type IIB restriction-modification systems, such as BcgI, feature a single protein with both endonuclease and methyltransferase activities. Type IIB nucleases require two recognition sites and cut both strands on both sides of their unmodified sites. BcgI cuts all eight target phosphodiester bonds before dissociation. The BcgI protein contains A and B polypeptides in a 2:1 ratio: A has one catalytic centre for each activity; B recognizes the DNA. We show here that BcgI is organized as A2B protomers, with B at its centre, but that these protomers self-associate to assemblies containing several A2B units. Moreover, like the well known FokI nuclease, BcgI bound to its site has to recruit additional protomers before it can cut DNA. DNA-bound BcgI can alternatively be activated by excess A subunits, much like the activation of FokI by its catalytic domain. Eight A subunits, each with one centre for nuclease activity, are presumably needed to cut the eight bonds cleaved by BcgI. Its nuclease reaction may thus involve two A2B units, each bound to a recognition site, with two more A2B units bridging the complexes by protein–protein interactions between the nuclease domains. PMID:23147005
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.
Literatura expandida: el fenómeno fanfiction en español

OpenAIRE

Sanz Martínez, Sandra

2017-01-01

En este trabajo se pretende abordar el estudio del llamado “fenómeno fanfiction”, un fenómeno que ha sacudido internet en las últimas décadas y que supone una nueva forma de creación de literatura. La creación de fanfiction supone la realización de una obra, normalmente narrativa, cuyo contenido contiene aspectos de una obra de ficción publicada, siempre sin ánimo de lucro. Departamento de Literatura Española y Teoría de la Literatura y Literatura Comparada Grado en Español: Lengua y...
The γ-gliadin-like γ-prolamin genes in the tribe Triticeae

Indian Academy of Sciences (India)

Supplementary data: The γ-gliadin-like γ-prolamin genes in the tribe Triticeae. Peng-Fei Qi, Cheng-Xing Le, Zhao Wang, Yu-Bin Liu, Qing Chen, Zhen-Zhen Wei, Bin-Jie Xu, Zheng-Yuan Wei,. Shou-Fen Dai, Yu-Ming Wei and You-Liang Zheng. J. Genet. 93, 35–41. Table 1. The γ-prolamin genes of diploid Triticeae species.
Fenótipos clínicos de asma grave Clinical phenotypes of severe asthma

Directory of Open Access Journals (Sweden)

Roseliane de Souza Araújo Alves

2008-09-01

Full Text Available OBJETIVO: Estabelecer os fenótipos clínicos em portadores de asma grave. MÉTODOS: Foram estudados, retrospectivamente, 111 pacientes em um ambulatório especializado. Os pacientes foram avaliados e acompanhados de maneira sistemática, estabelecendo-se ao final do acompanhamento a adesão e o controle ou não da doença por dados clínicos e funcionais. A resistência ao tratamento foi definida como o não preenchimento, ao final do acompanhamento, por pelo menos seis meses, dos critérios de controle de asma, apesar do uso correto e adesão à medicação. Os fenótipos foram determinados por análise fatorial e comparados por testes diversos. RESULTADOS: Ao final, 88 pacientes foram considerados aderentes e 23 não aderentes. Por análise fatorial do grupo aderente, quatro fenótipos foram determinados: o fenótipo 1 (28 pacientes, formado pelos pacientes resistentes ao tratamento, com maior freqüência de sintomas noturnos, maior número de exacerbações e uso mais freqüente de broncodilatador de resgate; o fenótipo 2 (48 pacientes, formado pelos pacientes com obstrução persistente, com menores valores de relação volume expiratório forçado no primeiro segundo/capacidade vital forçada na avaliação inicial, idade mais avançada e maior tempo de doença; o fenótipo 3 (42 pacientes, representa os pacientes com rinossinusite alérgica, sendo constituído de não fumantes com obstrução predominantemente reversível; e o fenótipo 4 (15 pacientes, formado por casos com história de intolerância à aspirina associado à asma quase fatal. CONCLUSÕES: Um número significativo de portadores de ama grave não adere ao tratamento. Muitos pacientes com asma grave têm obstrução irreversível, mas o fenótipo clínico mais relevante é constituído pelos pacientes resistentes ao tratamento habitual.OBJECTIVE: To characterize clinical phenotypes of severe asthma. METHODS: A total of 111 patients were retrospectively evaluated at a
Free energy landscape of electrocatalytic CO2 reduction to CO on aqueous FeN4 center embedded graphene studied by ab initio molecular dynamics simulations

Science.gov (United States)

Sheng, Tian; Sun, Shi-Gang

2017-11-01

Experiments have found that the porphyrin-like FeN4 site in Fe-N-C materials is highly efficient for the electrochemical reduction of CO2 into CO. In this work, we investigated the reduction mechanisms on FeN4 embedded graphene layer catalyst with some explicit water molecules by combining the constrained ab initio molecular dynamics simulations and thermodynamic integrations. The reaction free energy and electron transfer in each elementary step were identified. The initial CO2 activation was identified to go through the first electron transfer to form adsorbed CO2- anion and the CO desorption was the rate limiting step in the overall catalytic cycle.
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.
Cholesterol-Containing Nuclease-Resistant siRNA Accumulates in Tumors in a Carrier-free Mode and Silences MDR1 Gene

Directory of Open Access Journals (Sweden)

Ivan V. Chernikov

2017-03-01

Full Text Available Chemical modifications are an effective way to improve the therapeutic properties of small interfering RNAs (siRNAs, making them more resistant to degradation in serum and ensuring their delivery to target cells and tissues. Here, we studied the carrier-free biodistribution and biological activity of a nuclease-resistant anti-MDR1 cholesterol-siRNA conjugate in healthy and tumor-bearing severe combined immune deficiency (SCID mice. The attachment of cholesterol to siRNA provided its efficient accumulation in the liver and in tumors, and reduced its retention in the kidneys after intravenous and intraperitoneal injection. The major part of cholesterol-siRNA after intramuscular and subcutaneous injections remained in the injection place. Confocal microscopy data demonstrated that cholesterol-siRNA spread deep in the tissue and was present in the cytoplasm of almost all the liver and tumor cells. The reduction of P-glycoprotein level in human KB-8-5 xenograft overexpressing the MDR1 gene by 60% was observed at days 5–6 after injection. Then, its initial level recovered by the eighth day. The data showed that, regardless of the mode of administration (intravenous, intraperitoneal, or peritumoral, cholesterol-siMDR efficiently reduced the P-glycoprotein level in tumors. The designed anti-MDR1 conjugate has potential as an adjuvant therapeutic for the reversal of multiple drug resistance of cancer cells.
Microbial ecology in a future climate: effects of temperature and moisture on microbial communities of two boreal fens.

Science.gov (United States)

Peltoniemi, Krista; Laiho, Raija; Juottonen, Heli; Kiikkilä, Oili; Mäkiranta, Päivi; Minkkinen, Kari; Pennanen, Taina; Penttilä, Timo; Sarjala, Tytti; Tuittila, Eeva-Stiina; Tuomivirta, Tero; Fritze, Hannu

2015-07-01

Impacts of warming with open-top chambers on microbial communities in wet conditions and in conditions resulting from moderate water-level drawdown (WLD) were studied across 0-50 cm depth in northern and southern boreal sedge fens. Warming alone decreased microbial biomass especially in the northern fen. Impact of warming on microbial PLFA and fungal ITS composition was more obvious in the northern fen and linked to moisture regime and sample depth. Fungal-specific PLFA increased in the surface peat in the drier regime and decreased in layers below 10 cm in the wet regime after warming. OTUs representing Tomentella and Lactarius were observed in drier regime and Mortierella in wet regime after warming in the northern fen. The ectomycorrhizal fungi responded only to WLD. Interestingly, warming together with WLD decreased archaeal 16S rRNA copy numbers in general, and fungal ITS copy numbers in the northern fen. Expectedly, many results indicated that microbial response on warming may be linked to the moisture regime. Results indicated that microbial community in the northern fen representing Arctic soils would be more sensitive to environmental changes. The response to future climate change clearly may vary even within a habitat type, exemplified here by boreal sedge fen. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
E-sports: um fenómeno da cultura digital contemporânea

OpenAIRE

Saraiva, Pedro André Cardoso

2013-01-01

Mestrado em Comunicação, Cultura e Tecnologias de Informação A competição entre dois jogadores está na génese dos jogos digitais e é esse o fator que está no centro deste trabalho, que explora o fenómeno do desporto eletrónico, os e-sports. Este conceito representa a prática competitiva e profissionalmente organizada nos videojogos. Situado conceptualmente entre o gaming e o desporto, os e-sports apresentam-se como um fenómeno da cultura digital contemporânea, partic...
Fenómenos mixtos en la división neurosis y psicosis: un estudio de las elaboraciones freudianas

OpenAIRE

Aguirre, Javier

2013-01-01

El presente trabajo aborda algunos desarrollos freudianos referidos a la división neurosis y psicosis. El análisis se centra en la hipótesis referida a la existencia de fenómenos mixtos, neuróticos y psicóticos, o sea, fenómenos que se presentarían de una misma manera en su articulación en ambas categorías. Se concluye que la expresión “fenómenos mixtos” no es adecuada, en la medida que Freud aborda el fenómeno siempre de una manera articulada y no de una manera aislada, es decir, la caracter...
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.
The BMP antagonist follistatin-like 1 is required for skeletal and lung organogenesis

NARCIS (Netherlands)

Sylva, M.; Li, V.S.; Buffing, A.A.; van Es, J.H.; van den Born, M.M.W.; van der Velden, S.; Gunst, Q.; Koolstra, J.H.; Moorman, A.F.; Clevers, H.; van den Hoff, M.J.

2011-01-01

Follistatin-like 1 (Fstl1) is a secreted protein of the BMP inhibitor class. During development, expression of Fstl1 is already found in cleavage stage embryos and becomes gradually restricted to mesenchymal elements of most organs during subsequent development. Knock down experiments in chicken and
The receptor-like kinase SOBIR1 interacts with Brassica napus LepR3 and is required for Leptosphaeria maculans AvrLm1-triggered immunity

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Lisong eMa

2015-10-01

Full Text Available AbstractThe fungus Leptosphaeria maculans (L. maculans is the causal agent of blackleg disease of canola/oilseed rape (Brassica napus worldwide. We previously reported cloning of the B. napus blackleg resistance gene, LepR3, which encodes a receptor-like protein. LepR3 triggers localised cell death upon recognition of its cognate Avr protein, AvrLm1. Here, we exploited the Nicotiana benthamiana model plant to investigate the recognition mechanism of AvrLm1 by LepR3. Co-expression of the LepR3/AvrLm1 gene pair in N. benthamiana resulted in development of a hypersensitive response (HR. However, a truncated AvrLm1 lacking its indigenous signal peptide was compromised in its ability to induce LepR3-mediated HR, indicating that AvrLm1 is perceived by LepR3 extracellularly. Structure-function analysis of the AvrLm1 protein revealed that the C-terminal region of AvrLm1 was required for LepR3-mediated HR in N. benthamiana and for resistance to L. maculans in B. napus. LepR3 was shown to be physically interacting with the B. napus receptor like kinase, SOBIR1 (BnSOBIR1. Silencing of NbSOBIR1 or NbSERK3 (BAK1 compromised LepR3-AvrLm1-dependent HR in N. benthamiana, suggesting that LepR3-mediated resistance to L. maculans in B. napus requires SOBIR1 and BAK1/SERK3. Using this model system, we determined that BnSOBIR1 and SERK3/BAK1 are essential partners in the LepR3 signalling complex and were able to define the AvrLm1 effector domain.
Measurement of 54Fe(n,2n)53Fe cross section near threshold

International Nuclear Information System (INIS)

Smither, R.K.; Greenwood, L.R.

1984-01-01

A series of experiments were performed at the Princeton Plasma Physics Laboratory to measure the cross section of the 54 Fe(n,2n) 53 Fe reaction near threshold. Measurements were made at 6 different neutron energies and cover the 1 MeV energy range from threshold (13.64 MeV) to 14.64 MeV. The 54 Fe(n,2n) cross section was measured relative to the 27 Al(n,p) 27 Mg cross section to an accuracy of a few percent. These accurate cross-section measurements will be useful in calculating damage caused by 14 MeV D-T plasma neutrons in Fe and calculating the production of the long-lived 53 Mn nuclei that account for much of the buildup of long-lived radioactivity in steel structures and other ferrous materials used in the construction of fusion reactors. They will also play an important part in a new method for measuring the plasma ion temperature of a D-T plasma

Una revisión del fenómeno de aislamiento magnético

Directory of Open Access Journals (Sweden)

Zósimo Arévalo Velosa

2007-09-01

Full Text Available Los dispositivos de alta energía son diseñados para que funcionen con campos eléctricos y magnéticos extrema-damente altos. Debido a esto, dichos dispositivos presentan fenómenos y comportamientos no lineales, como el aislamiento magnético que altera el transporte de los electrones. La no linealidad ha obligado a analizar y revisar desde el punto de vista matemático las condiciones de frontera y las soluciones del problema. El objetivo de este trabajo es exponer el tema a un nivel que facilite su difusión entre la comunidad académica no familiarizada con el mismo. Para ello, el trabajo presenta una descripción de los fenómenos que se generan cuando hay campos eléctricos y magnéticos muy altos, tomando el caso de un diodo plano al vacío, así como las ecuaciones que modelan el fenómeno de aislamiento magnético; también demuestra la existencia de las soluciones y encuentra las positivas en base a los métodos de solución superior e inferior para problemas de valor de frontera, y pro-porciona algunos ejemplos de aplicación del fenómeno de aislamiento magnético.
Annual cycle of methane emission from a boreal fen measured by the eddy covariance technique

International Nuclear Information System (INIS)

Rinne, Janne.; Pihlatie, Mari; Haapanala, Sami; Vesala, Timo; Riutta, Terhi; Tuittila, Eeva-Stiina; Aurela, Mika; Tuovinen, Juha-Pekka

2007-01-01

The northern wetlands are one of the major sources of methane into the atmosphere. We measured annual methane emission from a boreal minerotrophic fen, Siikaneva, by the eddy covariance method. The average wintertime emissions were below 1 mg/m 2 /h, and the summertime emissions about 3.5 mg/m 2 /h. The water table depth did have any clear effect on methane emissions. During most of the year the emission depended on the temperature of peat below the water table. However, during the high and late summer the emission was independent on peat temperature as well. No diurnal cycle of methane flux was found. The total annual emission from the Siikaneva site was 12.6 g/m 2 . The emissions of the snow free period contributed 91% to the annual emission. The emission pulse during the snow melting period was clearly detectable but of minor importance adding only less than 3% to the annual emission. Over 20% of the carbon assimilated during the year as carbon dioxide was emitted as methane. Thus methane emission is an important component of the carbon balance of the Siikaneva fen. This indicates need of taking methane into account when studying carbon balances of northern fen ecosystems
Filtering fens: Mechanisms explaining phosphorus-limited hotspots of biodiversity in wetlands adjacent to heavily fertilized areas

NARCIS (Netherlands)

Cusell, C.; Kooijman, A.; Fernandez, F.; van Wirdum, G.; Geurts, J.J.M.; van Loon, E.E.; Kalbitz, K.; Lamers, L.P.M.

2014-01-01

The conservation of biodiverse wetland vegetation, including that of rich fens, has a high priority at a global scale. Although P-eutrophication may strongly decrease biodiversity in rich fens, some well-developed habitats do still survive in highly fertilized regions due to nutrient filtering
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
Contrasting species-environment relationships in communities of testate amoebae, bryophytes and vascular plants along the fen-bog gradient.

Science.gov (United States)

Lamentowicz, Mariusz; Lamentowicz, Lukasz; van der Knaap, Willem O; Gabka, Maciej; Mitchell, Edward A D

2010-04-01

We studied the vegetation, testate amoebae and abiotic variables (depth of the water table, pH, electrical conductivity, Ca and Mg concentrations of water extracted from mosses) along the bog to extremely rich fen gradient in sub-alpine peatlands of the Upper Engadine (Swiss Alps). Testate amoeba diversity was correlated to that of mosses but not of vascular plants. Diversity peaked in rich fen for testate amoebae and in extremely rich fen for mosses, while for testate amoebae and mosses it was lowest in bog but for vascular plants in extremely rich fen. Multiple factor and redundancy analyses (RDA) revealed a stronger correlation of testate amoebae than of vegetation to water table and hydrochemical variables and relatively strong correlation between testate amoeba and moss community data. In RDA, hydrochemical variables explained a higher proportion of the testate amoeba and moss data than water table depth. Abiotic variables explained a higher percentage of the species data for testate amoebae (30.3% or 19.5% for binary data) than for mosses (13.4%) and vascular plants (10%). These results show that (1) vascular plant, moss and testate amoeba communities respond differently to ecological gradients in peatlands and (2) testate amoebae are more strongly related than vascular plants to the abiotic factors at the mire surface. These differences are related to vertical trophic gradients and associated niche differentiation.
Evaluation on the Efficiency of Subsurface Drainage in Chiu-Fen Landslide at Northern Taiwan

Science.gov (United States)

Ying, L. Y.; Lin, D. G.

2015-12-01

For administrative district, the Chiu-Fen landslide is situated at northern Taiwan and comes within the jurisdiction of Ruei-Fang district, New Taipei City Government. Chiu-Fen village is a famous spot for sightseeing and tourism in Southeast Asia. In the last decade, for economic purpose, a vast area of slope land in Chiu-Fen area was reclaimed into business and commercial districts. However, due to the complicated geological and hydrological conditions, improper reclamation, and lack of appropriate soil and water conservation facilities, large scale landslides are frequently triggered by typhoon rainfall and causes damages to the transportation and residential building in the community. As a consequence, the government initiated a comprehensive field investigations and remediation plans to stabilize the landslide from 1997 and the remediation works were concentrated on subsurface drainages, namely the application of drainage well (a vertical shaft with multi-level horizontal drainage boreholes). To investigate the efficiency of drainage wells on the landslide, the A1-profile in the landslide which covers the drainage wells W2 and W4 was selected for a series of rainfall seepage and slope stability analyses. In addition, a 48-hrs design rainfall with return period of 25, 50 and 100 years based on the local meteorological data bank was adopted for the analyses. The numerical results indicate the factor safety FS of the three potential sliding surfaces within A1-profile are constantly keeping greater than one (FS > 1.0) and without decreasing with the elapsed time during rainfall. This implies that the subsurface drainage works can drain off the infiltrated rainwater from a high intensity and long duration rainfall and preserve the slope stability of landslides from deterioration. Finally, the efficiency of the drainage wells can be evaluated quantitatively in terms of the time-dependent factor of safety and the pore water pressure distribution on several potential
A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering

Science.gov (United States)

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

2013-01-01

Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA binding proteins found in the plant pathogen Xanthomonas sp. The DNA binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within one week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using, respectively, qRT-PCR and Surveyor nuclease. The TALE toolbox described here will enable a broad range of biological applications. PMID:22222791
Metódica: Articulación sistema teórico, fenómeno y procesos en el objeto de estudio. Una perspectiva desde el fenómeno pobreza

OpenAIRE

Yajaira Jiménez Zabala

2011-01-01

En este artículo discutimos la visión metódica que toma como apoyo el pivote entre lo cualitativo, lo cuantitativo y el orden de complejidad, en un enfoque plurimetódico, a través del cual se construyó la visión de la pobreza como objeto de estudio. Tal selección, está justificada en tanto se entiende la pobreza como un fenómeno social complejo, por las diversas aristas que le identifican. La combinación de los enfoques metodológicos permitió la aprehensión de los diferentes aspectos del fenó...
Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula

Science.gov (United States)

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
Experimental acidification of a poor fen in northwestern Ontario: Effects on emergence of Chironomidae (Diptera)

Energy Technology Data Exchange (ETDEWEB)

Rosenberg, D.M.; Wiens, A.P.; Bilyj, B. [Department of Fisheries and Oceans, Winnipeg, MB (Canada). Central and Arctic Region; Armstrong, L. [Department of Fisheries and Oceans, Winnipeg, MB (Canada). Central and Arctic Region

1995-10-01

The effects of experimental acidification and N fertilization on chironomid emergence from a poor fen in northwestern Ontario were studied from 1984 to 1989. Experimental treatments did not affect the numbers of chironomids emerging weekly, the timing of emergence, or the species composition of emergence during the study period. Weekly emergence counts were higher (P<0.05) from the periphery of the fen than the central part. Total seasonal emergence was higher from an area that was previously burned than from the rest of that swamp, though the difference was not statistically significant. Stability of species composition, despite experimental treatments and physiographic differences in the fen, may be a widespread characteristic of Canadian acidic peatlands. 7 tabs., 4 figs., 23 refs.
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.
Fe-N co-doped SiO2@TiO2 yolk-shell hollow nanospheres with enhanced visible light photocatalytic degradation

Science.gov (United States)

Wan, Hengcheng; Yao, Weitang; Zhu, Wenkun; Tang, Yi; Ge, Huilin; Shi, Xiaozhong; Duan, Tao

2018-06-01

SiO2@TiO2 yolk@shell hollow nanospheres (STNSs) is considered as an outstanding photocatalyst due to its tunable structure and composition. Based on this point, we present an unprecedentedly excellent photocatalytic property of STNSs toward tannic acid via a Fe-N co-doped strategy. Their morphologies, compositions, structure and properties are characterized. The Fe-N co-doped STNSs formed good hollow yolk@shell structure. The results show that the energy gap of the composites can be downgraded to 2.82 eV (pure TiO2 = 3.2 eV). Photocatalytic degradation of tannic acid (TA, 30 mg L-1) under visible light (380 nm TiO2 nanospheres, non-doped STNSs and N-doped STNSs, the Fe-N co-doped STNSs exhibits the highest activity, which can degrade 99.5% TA into CO2 and H2O in 80 min. The probable degradation mechanism of the composites is simultaneously proposed, the band gap of STNSs becomes narrow by co-doping Fe-N, so that the TiO2 shell can stimulate electrons under visible light exposure, generate the ions of radOH and radO2- with a strong oxidizing property. Therefore this approach works is much desired for radioactive organic wastewater photocatalytic degradation.
Formation dynamics of FeN thin films on Cu(100)

KAUST Repository

Heryadi, Dodi; Schwingenschlö gl, Udo

2012-01-01

To investigate the structural and magnetic properties of thin films of FeN we have performed ab initio molecular dynamics simulations of their formation on Cu(100) substrates. The iron nitride layers exhibit a p4gm(2 × 2) reconstruction and order
Restoring sedges and mosses into frost heaving iron fens, San Juan Mountains, Colorado

Directory of Open Access Journals (Sweden)

R.A. Chimner

2011-08-01

Full Text Available Rare iron fens in the San Juan Mountains of Colorado are frequently in poor condition due to mining, roads and ditches, which have left much of the fen completely bare of vegetation. Natural revegetation is slow to occur in the bare areas because of severe frost heave in the cold mountain climate. Therefore, experimental revegetation plots were conducted in a factorial design with mulching and no mulching, crossed with moss diaspores, sedge transplants, and moss and sedge combined. Mulching influenced surface soil temperatures by reducing the midday highs and increasing the night-time lows, which decreased the frequency and amount of frost heave. Peat moisture also modified frost heave, with the greatest frost heaving occurring near 75 % peat moisture content (water table 10–20 cm below the surface and the least when soils were either wetter or drier. Moss survival was dependent on mulch, with no moss surviving in plots without mulch. Mulching also increased sedge transplant survival. In summary, mulching significantly increased the success of vegetation restoration efforts for frost heave areas in mountain fens.
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
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
Violencia en la pareja, las caras del fenómeno

Directory of Open Access Journals (Sweden)

Luz Elena Ocampo Otálvaro

2011-01-01

Full Text Available Este trabajo pretende mostrar las diversas caras o facetas del fenómeno de la violencia en la pareja, el mismo que en la literatura es conocido como violencia conyugal, violencia doméstica, violencia por parte del compañero íntimo, etc. Se identifica como un problema de salud pública, debido a los altos costos que genera y la manera como lesiona la vida cotidiana de quienes lo padecen. Se manifiesta en el seno de las relaciones de pareja, e irradia hacia los diversos ámbitos de interacción de los actores: mujer, varón, niño, niña. Involucra a todo el grupo o los grupos familiares de quienes lo viven, y puede lesionar de forma severa a los mismos. Se revisan aspectos del fenómeno, como el hecho que no sólo se presenta en las relaciones en las que la pareja tenga una unión de hecho, sino también en parejas de novios. No solo afecta a las mujeres o a las niñas; también afecta a los hombres. Tampoco es exclusivo de las parejas heterosexuales; los estudios muestran que entre parejas del mismo sexo también está presente. Se trata entonces de un fenómeno universal, multicausal, multidimensional, de salud y de tipo legal, que genera consecuencias que incluyen desde un problema de autoestima, de ansiedad, de depresión, de estrés hasta la muerte misma.
A Genetic Screen Identifies a Requirement for Cysteine-Rich-Receptor-Like Kinases in Rice NH1 (OsNPR1-Mediated Immunity.

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Mawsheng Chern

2016-05-01

Full Text Available Systemic acquired resistance, mediated by the Arabidopsis NPR1 gene and the rice NH1 gene, confers broad-spectrum immunity to diverse pathogens. NPR1 and NH1 interact with TGA transcription factors to activate downstream defense genes. Despite the importance of this defense response, the signaling components downstream of NPR1/NH1 and TGA proteins are poorly defined. Here we report the identification of a rice mutant, snim1, which suppresses NH1-mediated immunity and demonstrate that two genes encoding previously uncharacterized cysteine-rich-receptor-like kinases (CRK6 and CRK10, complement the snim1 mutant phenotype. Silencing of CRK6 and CRK10 genes individually in the parental genetic background recreates the snim1 phenotype. We identified a rice mutant in the Kitaake genetic background with a frameshift mutation in crk10; this mutant also displays a compromised immune response highlighting the important role of crk10. We also show that elevated levels of NH1 expression lead to enhanced CRK10 expression and that the rice TGA2.1 protein binds to the CRK10 promoter. These experiments demonstrate a requirement for CRKs in NH1-mediated immunity and establish a molecular link between NH1 and induction of CRK10 expression.
CO dissociation on magnetic Fen clusters

KAUST Repository

Jedidi, Abdesslem

2014-01-01

This work theoretically investigates the CO dissociation on Fen nanoparticles, for n in the range of 1-65, focusing on size dependence in the context of the initial step of the Fischer-Tropsch reaction. CO adsorbs molecularly through its C-end on a triangular facet of the nanoparticle. Dissociation becomes easier when the cluster size increases. Then, the C atom is bonded to a square facet that is generated as a result of the adsorption if it does not yet exist in the bare cluster, while the O atom is adsorbed on a triangular facet. In the most stable situation, the two adsorbed atoms remain close together, both having in common one shared first-neighbor iron atom. There is a partial spin quenching of the neighboring Fe atoms, which become more positively charged than the other Fe atoms. The shared surface iron atom resembles a metal-cation from a complex. Despite the small size of the iron cluster considered, fluctuations due to specific configurations do not influence properties for n > 25 and global trends seem significant.

Mung Bean nuclease mapping of RNAs 3' end

Directory of Open Access Journals (Sweden)

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.
Compartmentalised expression of Delta-like 1 in epithelial somites is required for the formation of intervertebral joints

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Gossler Achim

2007-06-01

Full Text Available Abstract Background Expression of the mouse Delta-like 1 (Dll1 gene in the presomitic mesoderm and in the caudal halves of somites of the developing embryo is required for the formation of epithelial somites and for the maintenance of caudal somite identity, respectively. The rostro-caudal polarity of somites is initiated early on within the presomitic mesoderm in nascent somites. Here we have investigated the requirement of restricted Dll1 expression in caudal somite compartments for the maintenance of rostro-caudal somite polarity and the morphogenesis of the axial skeleton. We did this by overexpressing a functional copy of the Dll1 gene throughout the paraxial mesoderm, in particular in anterior somite compartments, during somitogenesis in transgenic mice. Results Epithelial somites were generated normally and appeared histologically normal in embryos of two independent Dll1 over-expressing transgenic lines. Gene expression analyses of rostro-caudal marker genes suggested that over-expression of Dll1 without restriction to caudal compartments was not sufficient to confer caudal identity to rostral somite halves in transgenic embryos. Nevertheless, Dll1 over-expression caused dysmorphologies of the axial skeleton, in particular, in morphological structures that derive from the articular joint forming compartment of vertebrae. Accordingly, transgenic animals exhibited missing or reduced intervertebral discs, rostral and caudal articular processes as well as costal heads of ribs. In addition, the midline of the vertebral column did not develop normally. Transgenic mice had open neural arches and split vertebral bodies with ectopic pseudo-growth plates. Endochondral bone formation and ossification in the developing vertebrae were delayed. Conclusion The mice overexpressing Dll1 exhibit skeletal dysmorphologies that are also evident in several mutant mice with defects in somite compartmentalisation. The Dll1 transgenic mice demonstrate that
Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis.

Science.gov (United States)

Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura; Lees-Miller, Susan P

2015-08-01

Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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
Emissions of biogenic sulfur gases from northern bogs and fens

Science.gov (United States)

Demello, William Zamboni; Hines, Mark E.; Bayley, Suzanne E.

1992-01-01

Sulfur gases are important components of the global cycle of S. They contribute to the acidity of precipitation and they influence global radiation balance and climate. The role of terrestrial sources of biogenic S and their effect on atmospheric chemistry remain as major unanswered questions in our understanding of the natural S cycle. The role of northern wetlands as sources and sinks of gaseous S by measuring rates of S gas exchange as a function of season, hydrologic conditions, and gradients in tropic status was investigated. Experiments were conducted in wetlands in New Hampshire, particularly a poor fen, and in Mire 239, a poor fen at the Experimental Lakes Area (ELA) in Ontario. Emissions were determined using Teflon enclosures, gas cryotrapping methods and gas chromatography (GC) with flame photometric detection. Dynamic (sweep flow) and static enclosures were employed which yielded similar results. Dissolved S gases and methane were determined by gas stripping followed by GC.
Comparisons of soil nitrogen mass balances for an ombrotrophic bog and a minerotrophic fen in northern Minnesota

Science.gov (United States)

Brian H. Hill; Terri M. Jicha; LaRae L.P. Lehto; Colleen M. Elonen; Stephen D. Sebestyen; Randy Kolka

2016-01-01

Wecompared nitrogen (N) storage and flux in soils froman ombrotrophic bogwith that of a minerotrophic fen to quantify the differences in N cycling between these two peatlands types in northernMinnesota (USA). Precipitation, atmospheric deposition, and bog and fen outflowswere analyzed for nitrogen species. Upland and peatland soil sampleswere analyzed for N content,...
Bartonellosis y fenómeno de Mooser

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Hernando Groot

1941-02-01

Full Text Available Es conocido el fenómeno o reacción de Mooser que este investigador lograra al inocular a un curí por vía intraperitoneal sangre de un tifoso exantemático mejicano, y que consiste en una edema escroto-testicular bastante típico (1-6. En líneas generales se puede decir que un curí inoculado intraperitonealmente con sangre de un tifoso exantemático, presenta después de un período de incubación que varía entre dos días y dos semanas (variación que depende especialmente de las dosis inoculadas uan fiebre de 39,8 a 40,5 que puede durar de 3 a 14 días. Clínicamente la hembra muestra sólo la reacción febril, mientras que en el macho se observan lesiones inflamatorias escroto-testiculares características.
Caracterização fenólica das uvas 'Alicante' e 'Syrah' cultivadas em safra fora de época

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Alessandro Jefferson Sato

2012-03-01

Full Text Available O objetivo do trabalho foi caracterizar a evolução da maturação fenólica das videiras 'Alicante' e 'Syrah' (Vitis vinifera L. cultivadas fora de época. A área experimental foi instalada em uma propriedade pertencente à Vinícola Intervin®, localizada em Maringá-PR. Os vinhedos foram estabelecidos em julho de 2001, e as plantas foram conduzidas no sistema latada (4,0 x 1,5 m, enxertadas sobre o porta-enxerto IAC 766 'Campinas'. As podas curtas de formação (duas gemas foram realizadas no fim do mês de agosto de 2007 e 2008, e as podas longas de produção (oito gemas foram realizadas no fim de fevereiro de 2008 e 2009. As avaliações foram realizadas em duas safras fora de época (2008 e 2009, a partir do início da maturação das uvas até sete dias após a colheita. Foram utilizadas 20 plantas representativas de cada variedade. Para a evolução da maturação fenólica das uvas, foi determinado o teor de antocianinas e o índice de polifenóis totais (IPT do mosto das bagas. Por ocasião da colheita, foi avaliado o teor de resveratrol do mosto das uvas. Para a 'Alicante', a média do teor de antocianinas foi de 55,4 mg 100g-1, IPT de 474,0 mg 100g-1 e teor de resveratrol de 7,6 µg g-1. Para a 'Syrah', observou-se teor médio de antocianinas de 44,8 mg 100g-1, IPT de 487,7 mg 100g-1 e teor de resveratrol de 6,4 µg g-1. Considera-se que a evolução da maturação fenólica das uvas estudadas neste trabalho (progressiva para antocianinas e oscilatória para IPT e o teor dos compostos fenólicos no momento da colheita são adequados para que estas sejam utilizadas como matéria-prima na elaboração de vinhos finos.
Geochemical response of a calcareous fen to road salt contamination during snow melt and precipitation events: Kampoosa Bog, Stockbridge, MA

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Rhodes, A. L.; Guswa, A. J.

2008-12-01

Kampoosa Bog is the largest and most diverse calcareous lake-basin fen remaining in Massachusetts, and it is one of the state's elite Areas of Critical Environmental Concern (ACEC). The ground water chemistry of the fen has been greatly altered by road salt runoff (NaCl) from the Massachusetts Turnpike, which crosses the northern margin of the wetland complex. Ground water samples collected at different depths within the wetland, measurements of exchangeable Na from an eight-meter core, and hydraulic conductivity measurements suggest that ground water flow and contamination is largely a near- surface phenomenon. Detailed sampling of surface and ground waters during three spring snow melt events and one precipitation event characterizes the geochemical response of the wetland to hydrologic events. Overall, Na:Cl ratios for surface and ground water samples are less than one, and sodium and chloride imbalances suggest that 20-30% of sodium from rock salt is stored on cation exchange sites on organic material. Na:Cl ratios greater than one for fen ground water sampled during Snow Melt 2007 suggest that sodium can be released from cation exchange sites back to ground water under dilute conditions. The total mass of Na and Cl exported from the wetland is greatest under conditions of high discharge. The flux of dissolved salts at the outlet of the fen during Snow Melt 2005 accounts for ~ 24% Na and ~ 32% Cl of rock salt added to the Massachusetts Turnpike during 2004-2005. Estimates of annual fluxes of Na and Cl are on par with the amount of road salt applied, and sodium and chloride concentrations in shallow groundwater have decreased since 2002. The months of March, April and May are the primary months for salt export, accounting for more than half of the annual salt flux in 2005. Concerning the annual net export of sodium and chloride, large rain events may be more important with removing dissolved salts from the fen than snow melt because snow melt also is a time when
Tolerância ao fenômeno alucinatório induzido pelo zolpidem: relato de caso Tolerancia al fenómeno alucinatorio inducido por el zolpidem: relato de caso Tolerance to zolpidem-induced hallucinations: case report

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Saint-Clair Bahls

2005-12-01

Full Text Available INTRODUÇÃO: O hipnótico zolpidem é considerado efetivo e seguro, entretanto, existem na literatura alguns relatos de caso de fenômeno alucinatório associado ao seu uso. Relataremos mais um caso de alucinações induzidas pelo zolpidem em paciente jovem do sexo feminino. DISCUSSÃO: Este caso, segundo nosso conhecimento, é o primeiro descrito na literatura que sugere a possibilidade de ocorrer, em alguns pacientes, o desenvolvimento de tolerância a esse efeito alucinatório adverso raro, porém, importante. Apresentamos uma discussão sobre algumas hipóteses farmacocinéticas e farmacodinâmicas deste fenômeno. CONCLUSÕES: Alertamos para a necessidade de atenção ao fato da indução alucinatória pelo zolpidem e sua tolerância, assim como a importância de seu melhor entendimento.INTRODUCTION: El hipnótico zolpidem es considerado efectivo y seguro. Sin embargo, hay en la literatura algunos relatos de caso de fenómeno alucinatorio asociado a su uso. Relataremos un caso más sobre alucinaciones inducidas por zolpidem en una paciente joven de sexo femenino. DISCUSIÓN: Este caso, según nuestro conocimiento, es el primero descrito en la literatura que sugiere la posibilidad de que en algunos pacientes ocurra el desarrollo de tolerancia a este efecto alucinatorio, adverso y raro, sin embargo, importante. Presentamos una discusión sobre algunas hipótesis farmacocinéticas y farmacodinámicas de este fenómeno. CONCLUSION: Por la necesidad de atención, alertamos al hecho de la inducción alucinatoria relacionada al zolpidem y a su tolerancia, así como a la importancia de comprenderla mejor.BACKGROUND: Zolpidem is reported to be a safe and effective hypnotic agent. In the literature, there are some case reports of hallucinations after zolpidem administration. We report one more case in witch zolpidem lead to hallucinations in young adult female. DISCUSSION: This case, to our knowledge, is the first to suggest the possibility of
Endogenous Glucagon-like Peptide-1 Receptor Signaling in the Nucleus Tractus Solitarius is Required for Food Intake Control.

Science.gov (United States)

Alhadeff, Amber L; Mergler, Blake D; Zimmer, Derek J; Turner, Christopher A; Reiner, David J; Schmidt, Heath D; Grill, Harvey J; Hayes, Matthew R

2017-06-01

Alhough the glucagon-like peptide-1 (GLP-1) system is critical to energy balance control and is a target for obesity pharmacotherapies, the receptor-population-mediating effects of endogenous GLP-1 signaling are not fully understood. To address this, we developed a novel adeno-associated virus (AAV-GLP-1R) that utilizes short hairpin RNA to chronically knock down GLP-1 receptors (GLP-1R) in rats. As pharmacological studies highlight the hindbrain nucleus tractus solitarius (NTS) as a brain region important for GLP-1R-mediated effects on energy balance, AAV-GLP-1R was injected into the NTS to examine the role of endogenous NTS GLP-1R signaling in energy balance control. Chow intake and meal size were significantly increased following chronic NTS GLP-1R knockdown. In addition, NTS GLP-1R knockdown significantly increased self-administration of palatable food under both fixed and progressive ratio schedules of reinforcement. Collectively, these data demonstrate that endogenous NTS GLP-1R signaling is required for the control of food intake and motivation to feed, and provide a new strategy to investigate the importance of distinct GLP-1R populations in the control of a variety of functions.
Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

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April Pawluk

2017-12-01

Full Text Available CRISPR (clustered regularly interspaced short palindromic repeat-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor.
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...
Changing Land Use: The Fens of England. A Case Study in Land Reclamation [And] Student Work Book.

Science.gov (United States)

Laws, Kevin

A social studies unit and student workbook explore changes in land use that have occurred in the Fenlands of England since the time it was first inhabited. Fens are lowlying land which is partially or completely covered with water. The English Fens are located on the eastern side of the British Isles and cover a total area of about 2,000 square…
Computerbasiert prüfen [Computer-based Assessment

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Frey, Peter

2006-08-01

Full Text Available [english] Computer-based testing in medical education offers new perspectives. Advantages are sequential or adaptive testing, integration of movies or sound, rapid feedback to candidates and management of web-based question banks. Computer-based testing can also be implemented in an OSCE examination. In e-learning environments formative self-assessment are often implemented and gives helpful feedbacks to learners. Disadvantages in high-stake exams are the high requirements as well for the quality of testing (e.g. standard setting as additionally for the information technology and especially for security. [german] Computerbasierte Prüfungen im Medizinstudium eröffnen neue Möglichkeiten. Vorteile solcher Prüfungen liegen im sequentiellen oder adaptiven Prüfen, in der Integration von Bewegtbildern oder Ton, der raschen Auswertung und zentraler Verwaltung der Prüfungsfragen via Internet. Ein Einsatzgebiet mit vertretbarem Aufwand sind Prüfungen mit mehreren Stationen wie beispielsweise die OSCE-Prüfung. Computerbasierte formative Selbsttests werden im Bereiche e-learning häufig angeboten. Das hilft den Lernenden ihren Wissensstand besser einzuschätzen oder sich mit den Leistungen anderer zu vergleichen. Grenzen zeigen sich bei den summativen Prüfungen beim Prüfungsort, da zuhause Betrug möglich ist. Höhere ärztliche Kompetenzen wie Untersuchungstechnik oder Kommunikation eigenen sich kaum für rechnergestützte Prüfungen.
Microbial Communities as Environmental Indicators of Ecological Disturbance in Restored Carbonate Fen-Results of 10 Years of Studies.

Science.gov (United States)

Mieczan, Tomasz; Tarkowska-Kukuryk, Monika

2017-08-01

Interactions between bacteria and protists are essential to the ecosystem ecology of fens. Until now, however, there has been almost no information on how restoration procedures in carbonate fens affect the functioning of microbial food webs. Changes in vegetation patterns resulting from restoration may take years to be observed, whereas microbial processes display effects even after short-term exposure to changes in environmental conditions caused by restoration. Therefore, microbial processes and patterns can be used as sensitive indicators of changes in environmental conditions. The present study attempts to verify the hypothesis that the species richness and abundance of microbial loop components would differ substantially before and after restoration. The effect of restoration processes on the functioning of the food web was investigated for a 10 years in a carbonate-rich fen, before and after restoration. The restoration procedure (particularly the improvement in hydrological conditions) distinctly modified the taxonomic composition and functioning of microbial food webs. This is reflected in the increased abundance and diversity of testate amoeba, i.e. top predators, within the microbial food web and in the pronounced increase in the abundance of bacteria. This study suggests potential use of microbial loop components as bio-indicators and bio-monitoring tools for hydrological status of fens and concentrations of nutrients. Better understanding of what regulates microbial populations and activity in fens and unravelling of these fundamental mechanisms are particularly critical in order to more accurately predict how fens will respond to global change or anthropogenic disturbances.
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...
Fen Bilgisi Öğretmen Adaylarının Kimya Bilgilerini Günlük Hayatla İlişkilendirebilme Düzeyleri

OpenAIRE

Yadigaroğlu, Yrd. Doç. Dr. Mustafa; DEMİRCİOĞLU, Doç. Dr. Gökhan; DEMİRCİOĞLU, Doç. Dr. Hülya

2017-01-01

Fen eğitiminin temel amaçlarındanbiri, öğrencilerin fen okur-yazarı olmalarını sağlayarak onları günlükyaşantılarında karşılaştıkları fen olaylarına anlam vermelerine yardımcıolmaktır. Günlük hayatla ya da bağlamı ile ilişkilendirilmeyen bilgi öğrencininzihninde her zaman eksik olacaktır. Bunun gerçekleştirilmesinde fen dersleriiçerisinde yer alan kimya önemli bir yere sahiptir. Bu düşünceden hareketleçalışmada, fen bilgisi öğretmen adaylarının kimya bilgilerini günlük hayatlailişkilendirebil...
N and P addition inhibits growth of rich fen bryophytes

DEFF Research Database (Denmark)

Andersen, Dagmar Kappel; Ejrnæs, Rasmus; Riis, Tenna

2016-01-01

vernicosus and paludella squarrosa) rich fen bryophytes were grown in mixed culture and subjected to rainwater or groundwater and three levels of N (0, 1 and 3 mg N L-1) and P (0, 0.05 and 0.1 mg P NL-1). All species responded negatively to higher N-levels and three of four species responded negatively...... to rainwater and higher P-levels. C. cuspidata had highest relative growth rate in all treatments, and the infrequently occurringrare species had lower relative growth rate and were more negatively affected by high levels of N than the frequently occurringcommon species. A negative effect of rainwater seemed...... to be caused by higher background levels of N in rainwater compared to groundwater rather than a pH-effect per se. We found a negative effect of high initial bryophyte density in three of four species indicating density dependent inhibition between species.We suggest that maintenance of oligotrophic conditions...
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.

Para Além do Estado e do Mercado: A Dádiva no Fenômeno dos Sem-Abrigo

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João Aldeia

2014-03-01

Full Text Available Las reflexiones sobre el fenómeno de los sin techo tienden a reproducir las ausencias que el paradigma del don identifica en el pensamiento sobre las sociedades modernas occidentales. Recurrentemente, se considera al Estado y al mercado como las únicas esferas de regulación social y se los interpreta como los espacios de donde derivan las causas estructurales y las posibles soluciones para el fenómeno. Ello no permite una comprensión completa del tema y tampoco una reflexión consistente sobre los modos de minimizar las injusticias de la vida en la calle. Pensar sobre el fenómeno por medio del paradigma del don es relevante porque permite identificar la habitual invisibilización de las relaciones interpersonales no oficiales que caracterizan la vida en la calle, posibilitando, a su vez, la revalorización de estas interacciones en el debate sobre el fenómeno de los sin techo.
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.
Gravidez na adolescência: um olhar sobre um fenômeno complexo

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Ana Cristina Garcia Dias

2010-04-01

Full Text Available O presente trabalho apresenta uma revisão seletiva e não sistemática da literatura a respeito do fenômeno da gestação na adolescência. Três eixos temáticos orientam a organização do trabalho: (a Riscos e problemas associados à gestação na adolescência; (b Fatores precursores relacionados à gravidez na adolescência; e (c Fatores sócio-culturais associados ao desejo de ser mãe na adolescência. Entre as conclusões, destaca-se que a gravidez na adolescência é uma experiência que pode ter consequências tanto negativas quanto positivas para os adolescentes. Além disso, o fenômeno evidencia a necessidade de intervenções voltadas à saúde sexual e reprodutiva dos adolescentes.
SLX-1 is required for maintaining genomic integrity and promoting meiotic noncrossovers in the Caenorhabditis elegans germline.

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Takamune T Saito

2012-08-01

Full Text Available Although the SLX4 complex, which includes structure-specific nucleases such as XPF, MUS81, and SLX1, plays important roles in the repair of several kinds of DNA damage, the function of SLX1 in the germline remains unknown. Here we characterized the endonuclease activities of the Caenorhabditis elegans SLX-1-HIM-18/SLX-4 complex co-purified from human 293T cells and determined SLX-1 germline function via analysis of slx-1(tm2644 mutants. SLX-1 shows a HIM-18/SLX-4-dependent endonuclease activity toward replication forks, 5'-flaps, and Holliday junctions. slx-1 mutants exhibit hypersensitivity to UV, nitrogen mustard, and camptothecin, but not gamma irradiation. Consistent with a role in DNA repair, recombination intermediates accumulate in both mitotic and meiotic germ cells in slx-1 mutants. Importantly, meiotic crossover distribution, but not crossover frequency, is altered on chromosomes in slx-1 mutants compared to wild type. This alteration is not due to changes in either the levels or distribution of double-strand breaks (DSBs along chromosomes. We propose that SLX-1 is required for repair at stalled or collapsed replication forks, interstrand crosslink repair, and nucleotide excision repair during mitosis. Moreover, we hypothesize that SLX-1 regulates the crossover landscape during meiosis by acting as a noncrossover-promoting factor in a subset of DSBs.
Los fenómenos psicológicos no investigados en la comunicación educativa

OpenAIRE

Ortiz-Torres, Emilio Alberto

2017-01-01

Propósito: el artículo tiene por objetivo valorar la importancia del estudio científico de los fenómenos psicológicos la sugestión, la percepción interpersonal y el rumor, los cuales inciden en la comunicación educativa. Tema: el tema que se trabaja es la existencia e influencia de dichos fenómenos subjetivos en los diferentes contextos formativos donde se desarrolla la comunicación interpersonal. Desarrollo: se realiza un análisis teórico sobre la comunicación educativa como concepto interdi...
Landscape history, calcareous fen development and historical events in the Slovak Eastern Carpathians

Czech Academy of Sciences Publication Activity Database

Jamrichová, Eva; Hájková, Petra; Horsák, M.; Rybníčková, E.; Lacina, A.; Hájek, Michal

2014-01-01

Roč. 23, č. 5 (2014), s. 497-513 ISSN 0939-6314 Institutional support: RVO:67985939 Keywords : forest development * spring fen * multi-proxy Subject RIV: EF - Botanics Impact factor: 2.648, year: 2014
Differential requirements for Tousled-like kinases 1 and 2 in mammalian development

DEFF Research Database (Denmark)

Segura-Bayona, Sandra; Knobel, Philip A.; Gonzalez-Buron, Helena

2017-01-01

, RNA interference, cell cycle progression, viral latency, chromosome segregation and mitosis. However, little is known about the functions of TLK activity in vivo or the relative functions of the highly similar TLK1 and TLK2 in any cell type. To begin to address this, we have generated Tlk1- and Tlk2......-deficient mice. We found that while TLK1 was dispensable for murine viability, TLK2 loss led to late embryonic lethality because of placental failure. TLK2 was required for normal trophoblast differentiation and the phosphorylation of ASF1 was reduced in placentas lacking TLK2. Conditional bypass...... of the placental phenotype allowed the generation of apparently healthy Tlk2-deficient mice, while only the depletion of both TLK1 and TLK2 led to extensive genomic instability, indicating that both activities contribute to genome maintenance. Our data identifies a specific role for TLK2 in placental function...
Fen ecohydrologic trajectories in response to groundwater drawdown with edaphic, floristic, and hydrologic feedbacks

Science.gov (United States)

Booth, E.; Steven, L. I.; Bart, D.

2017-12-01

Calcareous fens are unique and often isolated ecosystems of high conservation value worldwide because they provide habitat for many rare plant and animal species. Their identity is inextricably linked to an absolute dependence on a consistent discharge of groundwater that saturates the near surface for most of the growing season leading to the accumulation of carbon as peat or tufa and sequestration of nutrients. The stresses resulting from consistent saturation and low-nutrient availability result in high native plant diversity including very high rare species richness compared to other ecosystems. Decreases in the saturation stress by reduced groundwater inputs (e.g., from nearby pumping) can result in losses of native diversity, decreases in rare-species abundance, and increased invasion by non-native species. As such, fen ecosystems are particularly susceptible to changes in groundwater conditions including reduction in water levels due to nearby groundwater pumping. Trajectories of degradation are complex due to feedbacks between loss of soil organic carbon, changes in soil properties, and plant water use. We present a model of an archetype fen that couples a hydrological niche model with a variably-saturated groundwater flow model to predict changes in vegetation composition in response to different groundwater drawdown scenarios (step change, declining trend, and periodic drawdown during dry periods). The model also includes feedbacks among vegetation composition, plant water use, and soil properties. The hydrological niche models (using surface soil moisture as predictor) and relationships between vegetation composition, plant water use (via stomatal conductance and leaf-area index), and soil hydraulic properties (van Genuchten parameters) were determined based on data collected from six fens in Wisconsin under various states of degradation. Results reveal a complex response to drawdown and provide insight into other ecosystems with linkages between the
The effect of habitat fragmentation and abiotic factors on fen plant occurrence

NARCIS (Netherlands)

Soomers, H.; Karssenberg, D.J.; Verhoeven, J.T.A.; Verweij, P.A.; Wassen, M.J.

2013-01-01

Human landscape modification has led to habitat fragmentation for many species. Habitat fragmentation, leading to isolation, decrease in patch size and increased edge effect, is observed in fen ecosystems that comprise many endangered plant species. However, until now it has remained unclear
Fen (n=1–6) clusters chemisorbed on vacancy defects in graphene: Stability, spin-dipole moment, and magnetic anisotropy

KAUST Repository

Haldar, Soumyajyoti

2014-05-09

In this work, we have studied the chemical and magnetic interactions of Fen (n=1–6) clusters with vacancy defects (monovacancy to correlated vacancies with six missing C atoms) in a graphene sheet by ab initio density functional calculations combined with Hubbard U corrections for correlated Fe-d electrons. It is found that the vacancy formation energies are lowered in the presence of Fe, indicating an easier destruction of the graphene sheet. Due to strong chemical interactions between Fe clusters and vacancies, a complex distribution of magnetic moments appear on the distorted Fe clusters which results in reduced averaged magnetic moments compared to the free clusters. In addition to that, we have calculated spin-dipole moments and magnetic anisotropy energies. The calculated spin-dipole moments arising from anisotropic spin density distributions vary between positive and negative values, yielding increased or decreased effective moments. Depending on the cluster geometry, the easy axis of magnetization of the Fe clusters shows in-plane or out-of-plane behavior.
Rice Snl6, a cinnamoyl-CoA reductase-like gene family member, is required for NH1-mediated immunity to Xanthomonas oryzae pv. oryzae.

Directory of Open Access Journals (Sweden)

Rebecca S Bart

2010-09-01

Full Text Available Rice NH1 (NPR1 homolog 1 is a key mediator of innate immunity. In both plants and animals, the innate immune response is often accompanied by rapid cell death at the site of pathogen infection. Over-expression of NH1 in rice results in resistance to the bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo, constitutive expression of defense related genes and enhanced benzothiadiazole (BTH- mediated cell death. Here we describe a forward genetic screen that identified a suppressor of NH1-mediated lesion formation and resistance, snl6. Comparative genome hybridization and fine mapping rapidly identified the genomic location of the Snl6 gene. Snl6 is a member of the cinnamoyl-CoA reductase (CCR-like gene family. We show that Snl6 is required for NH1-mediated resistance to Xoo. Further, we show that Snl6 is required for pathogenesis-related gene expression. In contrast to previously described CCR family members, disruption of Snl6 does not result in an obvious morphologic phenotype. Snl6 mutants have reduced lignin content and increased sugar extractability, an important trait for the production of cellulosic biofuels. These results suggest the existence of a conserved group of CCR-like genes involved in the defense response, and with the potential to alter lignin content without affecting development.
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...
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
Modern distribution patterns of snails and plants in the Western Carpathian spring fens: is it a result of historical development?

Czech Academy of Sciences Publication Activity Database

Horsák, M.; Hájek, Michal; Dítě, D.; Tichý, L.

2007-01-01

Roč. 73, č. 1 (2007), s. 53-60 ISSN 0260-1230 Grant - others:GA AV ČR(CZ) KJB601630501; GA AV ČR(CZ) KJB6163302 Program:KJ Institutional research plan: CEZ:AV0Z60050516 Keywords : Snails * plants * rich fens Subject RIV: EF - Botanics Impact factor: 1.032, year: 2007
C. elegans VANG-1 modulates life span via insulin/IGF-1-like signaling.

Directory of Open Access Journals (Sweden)

Sebastian J Honnen

Full Text Available The planar cell polarity (PCP pathway is highly conserved from Drosophila to humans and a PCP-like pathway has recently been described in the nematode Caenorhabditis elegans. The developmental function of this pathway is to coordinate the orientation of cells or structures within the plane of an epithelium or to organize cell-cell intercalation required for correct morphogenesis. Here, we describe a novel role of VANG-1, the only C. elegans ortholog of the conserved PCP component Strabismus/Van Gogh. We show that two alleles of vang-1 and depletion of the protein by RNAi cause an increase of mean life span up to 40%. Consistent with the longevity phenotype vang-1 animals also show enhanced resistance to thermal- and oxidative stress and decreased lipofuscin accumulation. In addition, vang-1 mutants show defects like reduced brood size, decreased ovulation rate and prolonged reproductive span, which are also related to gerontogenes. The germline, but not the intestine or neurons, seems to be the primary site of vang-1 function. Life span extension in vang-1 mutants depends on the insulin/IGF-1-like receptor DAF-2 and DAF-16/FoxO transcription factor. RNAi against the phase II detoxification transcription factor SKN-1/Nrf2 also reduced vang-1 life span that might be explained by gradual inhibition of insulin/IGF-1-like signaling in vang-1. This is the first time that a key player of the PCP pathway is shown to be involved in the insulin/IGF-1-like signaling dependent modulation of life span in C. elegans.
The influence of vegetation structure on spider species richness, diversity and community organization in the Apšuciems calcareous fen, Latvia

Energy Technology Data Exchange (ETDEWEB)

Štokmane, M.; Spuņģis, V.

2016-07-01

Calcareous fens are considered to be among the most threatened ecosystems of Europe. They are also one of the most diverse habitats as they support an incredibly rich and diverse range of plant and animal species. However, in spite of their diversity, calcareous fens are still poorly investigated, especially when referring to fen invertebrates, such as spiders. Because spiders are good bioindicators, knowledge of their ecology in rare and threatened habitats is of interest. The aim of this study was to document the composition and diversity of spider species, families and foraging guilds in the ground– and grass–layers of the Apšuciems calcareous fen, and to evaluate the influence of vegetation structure on spider community organization. In summer 2012, we collected ground–dwelling spiders using pitfall traps and grass–dwelling spiders using sweep–netting. A total of 2,937 spider individuals belonging to 19 families and 80 species was collected in the Apšuciems fen. Our results indicate that spider species and families tend to be stratified across the vertical structure of the habitat; the spider composition in the ground stratum differed from that in the grass stratum. On the contrary, however, the spider foraging guild structure between the ground–layer and the grass–layer was similar. Each of the two studied strata presented similar guilds in similar proportions. Our results also showed that spider composition differed considerably between fen parts and that much of this variability could be explained by the architectural properties of the habitat. More diverse vegetation generally supported a higher number of spider species. (Author)
Análisis comparativo de compuestos fenólicos totales y actividad antioxidante de cuatro marcas de tisanas de Hibiscus sabdariffa (Malvaceae comercializadas en Costa Rica

Directory of Open Access Journals (Sweden)

Melissa Agüero

2014-01-01

Full Text Available Las tisanas de rosa de Jamaica (Hibiscus sabdariffa son consumidas en Costa Rica por su conocido efecto cardioprotector asociado a la presencia de compuestos fenólicos, principalmente antocianinas. Este trabajo estableció diferencias en el perfil de compuestos fenólicos y su actividad anti-radical entre diferentes comercializadores de tisanas de Hibiscus sabdariffa en Costa Rica. Se identificaron cualitativamente taninos, flavonoides y antocianinas. Se cuantificó la cantidad total de compuestos fenólicos por el método de Folin-Ciocalteu y se cuantificó la actividad antioxidante con el método de DPPH. Todas las marcas comerciales demostraron presencia de compuestos fenólicos, incluyendo antocianinas; sin embargo, se encontraron diferencias significativas en el contenido de compuestos fenólicos y actividad anti-radical, entre los diferentes productores de tisanas. Por ello, se concluye que en el mercado costarricense, el perfil en compuestos fenólicos de las tisanas en estudio varía de acuerdo con el productor.
Experiencias para observar el fenómeno de fluorescencia con luz ultravioleta

Directory of Open Access Journals (Sweden)

Heredia Avalos, S.

2008-09-01

Full Text Available La fluorescencia tiene lugar cuando una sustancia emite luz visible al absorber radiación de una longitud de onda menor, como por ejemplo luz ultravioleta. La diferencia entre la energía absorbida (del fotón ultravioleta y la emitida (del fotón visible se disipa en forma de calor. Se dice que la sustancia posee fluorescenciacuando este proceso es muy rápido; si es más lento se dice que la sustancia posee fosforescencia. A continuación se describen varias experiencias sencillas para poner de manifiesto el fenómeno de fluorescencia. Todas ellas requieren de reactivos y materiales fácilmente accesibles y asequibles y se pueden realizar con alumnos de secundaria o de bachillerato para poner de manifiesto este fenómeno. Además, también se propone una experiencia en la que se observa la reversibilidad de las reacciones químicas ácido-base a través de la fluorescencia.
Mycobacterium smegmatis RqlH defines a novel clade of bacterial RecQ-like DNA helicases with ATP-dependent 3'-5' translocase and duplex unwinding activities.

Science.gov (United States)

Ordonez, Heather; Unciuleac, Mihaela; Shuman, Stewart

2012-05-01

The Escherichia coli RecQ DNA helicase participates in a pathway of DNA repair that operates in parallel to the recombination pathway driven by the multisubunit helicase-nuclease machine RecBCD. The model mycobacterium Mycobacterium smegmatis executes homologous recombination in the absence of its helicase-nuclease machine AdnAB, though it lacks a homolog of E. coli RecQ. Here, we identify and characterize M. smegmatis RqlH, a RecQ-like helicase with a distinctive domain structure. The 691-amino acid RqlH polypeptide consists of a RecQ-like ATPase domain (amino acids 1-346) and tetracysteine zinc-binding domain (amino acids 435-499), separated by an RqlH-specific linker. RqlH lacks the C-terminal HRDC domain found in E. coli RecQ. Rather, the RqlH C-domain resembles bacterial ComF proteins and includes a phosphoribosyltransferase-like module. We show that RqlH is a DNA-dependent ATPase/dATPase that translocates 3'-5' on single-stranded DNA and has 3'-5' helicase activity. These functions inhere to RqlH-(1-505), a monomeric motor unit comprising the ATPase, linker and zinc-binding domains. RqlH homologs are distributed widely among bacterial taxa. The mycobacteria that encode RqlH lack a classical RecQ, though many other Actinobacteria have both RqlH and RecQ. Whereas E. coli K12 encodes RecQ but lacks a homolog of RqlH, other strains of E. coli have both RqlH and RecQ.
Compostos fenólicos totais, atividade antioxidante e antifúngica demultimisturas enriquecidas com a microalga Spirulina platensis

OpenAIRE

Bierhals, Vânia da Silva; Machado, Vanessa Goulart; Echevenguá, Walesca Oliveira; Costa, Jorge Alberto Vieira; Furlong, Eliana Badiale

2009-01-01

Com o objetivo de avaliar o conteúdo fenólico e as atividades antioxidantes e antifúngicas de multimisturas enriquecidas com Spirulina platensis, foram formuladas quatro multimisturas com farelo de arroz ou trigo, duas contendo Spirulina platensis e duas com sementes de girassol. Os compostos fenólicos foram estimados pela técnica de Folin-Ciocalteau. A atividade antioxidante foi avaliada por meio de inibição do radical livre DPPH e a atividade antifúngica foi analisada por meio de ensaio de ...

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1.

Science.gov (United States)

Yamano, Takashi; Zetsche, Bernd; Ishitani, Ryuichiro; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

2017-08-17

The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA) and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic genome editing. Cpf1 requires a specific nucleotide sequence, called a protospacer adjacent motif (PAM), for target recognition. Besides the canonical TTTV PAM, Cpf1 recognizes suboptimal C-containing PAMs. Here, we report four crystal structures of LbCpf1 in complex with the crRNA and its target DNA containing either TTTA, TCTA, TCCA, or CCCA as the PAM. These structures revealed that, depending on the PAM sequences, LbCpf1 undergoes conformational changes to form altered interactions with the PAM-containing DNA duplexes, thereby achieving the relaxed PAM recognition. Collectively, the present structures advance our mechanistic understanding of the PAM-dependent, crRNA-guided DNA cleavage by the Cpf1 family nucleases. Copyright © 2017 Elsevier Inc. All rights reserved.
Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

Science.gov (United States)

van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D.

2015-01-01

The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1–2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp. PMID:26507855
The influence of vegetation structure on spider species richness, diversity and community organization in the Apšuciems calcareous fen, Latvia

Directory of Open Access Journals (Sweden)

Štokmane, M.

2016-07-01

Full Text Available Calcareous fens are considered to be among the most threatened ecosystems of Europe. They are also one of the most diverse habitats as they support an incredibly rich and diverse range of plant and animal species. However, in spite of their diversity, calcareous fens are still poorly investigated, especially when referring to fen invertebrates, such as spiders. Because spiders are good bioindicators, knowledge of their ecology in rare and threatened habitats is of interest. The aim of this study was to document the composition and diversity of spider species, families and foraging guilds in the ground– and grass–layers of the Apšuciems calcareous fen, and to evaluate the influence of vegetation structure on spider community organization. In summer 2012, we collected ground–dwelling spiders using pitfall traps and grass–dwelling spiders using sweep–netting. A total of 2,937 spider individuals belonging to 19 families and 80 species was collected in the Apšuciems fen. Our results indicate that spider species and families tend to be stratified across the vertical structure of the habitat; the spider composition in the ground stratum differed from that in the grass stratum. On the contrary, however, the spider foraging guild structure between the ground–layer and the grass–layer was similar. Each of the two studied strata presented similar guilds in similar proportions. Our results also showed that spider composition differed considerably between fen parts and that much of this variability could be explained by the architectural properties of the habitat. More diverse vegetation generally supported a higher number of spider species.
Short-Term Summer Inundation as a Measure to Counteract Acidification in Rich Fens

NARCIS (Netherlands)

Mettrop, I.S.; Cusell, C.; Kooijman, A.M.; Lamers, L.P.M.

2015-01-01

In regions with intensive agriculture, water level fluctuation in wetlands has generally become constricted within narrow limits. Water authorities are, however, considering the re-establishment of fluctuating water levels as a management tool in biodiverse, base-rich fens (‘rich fens’). This
Nitrogen Dynamics Along a Headwater Stream Draining a Fen, Swamp, and Marsh in a Fractured Dolomite Watershed

Science.gov (United States)

Duval, T. P.; Waddington, J. M.

2009-05-01

Stream-wetland interaction has been shown to have a significant effect on nutrient cycling and downstream water quality. Additionally, connection to regional groundwater systems can dilute or enrich stream water with a number of dissolved constituents. This study demonstrates the resultant downstream change in dissolved nitrogen species as a hardwater stream emerges from a calcareous aquifer and traverses a calcareous fen, a cedar swamp, and a cattail marsh over two growing seasons, a very dry 2006 and a very wet 2007. Upon emergence at a number of groundwater seeps, the water contained appreciable nitrate levels averaging 2.72±0.42 mg NO3-N L-1, minimal organic nitrogen, and ammonium below detectable levels. Through the gently sloping calcareous fen, with a stream residence time of ~ 5 hours, NO3-N concentration decreases of 0.35 mg L-1 were observed. Concomitantly, stream recharge into the dolomite bedrock depressed stream discharge values significantly, further removing nitrate from the stream system. This resulted in the fen-bedrock system acting as an estimated net sink of 432 kg of NO3-N in the early summer of 2007, for example. In contrast, the hydrological-biogeochemical systems became decoupled through the swamp during the same period, where concentrations increased from 2.58±0.34 mg L-1 entering the swamp to 2.65±0.58 mg L-1 exiting, but streamflow decreased in general by 5 L s- 1. This resulted in the swamp, with its large depression storage, acting as a small net sink of nitrate (75 kg through the early summer), which would not be detected simply from concentration changes. The concentration-discharge relation realigned through the marsh, where significant groundwater entered the wetland, increasing both concentration and discharge, yielding a small export of 93 kg over the same time period. A series of tracer injections in each wetland type will be presented to compare the streamflow- concentration patterns with the measured nutrient spiralling
Eletricidade Atmosférica e Fenômenos Correlatos

OpenAIRE

Tavares, M.; Santiago, M.A.M

2002-01-01

A qualquer momento, existem cerca de 2000 tempestades com relâmpagos nos cêus em torno do mundo. Estima-se que as tempestades combinadas produzem aproximadamente 100 descargas de raios por segundo, cada uma com temperatura em torno de 30.0000ºC - mais quente que a superfície do Sol. Esse trabalho apresenta aos leitores alguns fenômenos elêtricos na atmosfera e estratosfera. Tais eventos fazem parte de um sugerido circuito elêtrico atmosfêrico global e estão conectados à pesquisa de plasmas. A...
Knockout of Myostatin by Zinc-finger Nuclease in Sheep Fibroblasts and Embryos

Directory of Open Access Journals (Sweden)

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.
Interacting effects of sulphate pollution, sulphide toxicity and eutrophication on vegetation development in fens: A mesocosm experiment

International Nuclear Information System (INIS)

Geurts, Jeroen J.M.; Sarneel, Judith M.; Willers, Bart J.C.; Roelofs, Jan G.M.; Verhoeven, Jos T.A.; Lamers, Leon P.M.

2009-01-01

Both eutrophication and SO 4 pollution can lead to higher availability of nutrients and potentially toxic compounds in wetlands. To unravel the interaction between the level of eutrophication and toxicity at species and community level, effects of SO 4 were tested in nutrient-poor and nutrient-rich fen mesocosms. Biomass production of aquatic and semi-aquatic macrophytes and colonization of the water layer increased after fertilization, leading to dominance of highly competitive species. SO 4 addition increased alkalinity and sulphide concentrations, leading to decomposition and additional eutrophication. SO 4 pollution and concomitant sulphide production considerably reduced biomass production and colonization, but macrophytes were less vulnerable in fertilized conditions. The experiment shows that competition between species, vegetation succession and terrestrialization are not only influenced by nutrient availability, but also by toxicity, which strongly interacts with the level of eutrophication. This implies that previously neutralized toxicity effects in eutrophied fens may appear after nutrient reduction measures have been taken. - Interspecific competition, vegetation succession and terrestrialization in fens depend on the interacting effects of SO 4 pollution, sulphide toxicity and nutrient availability.
Conversion of a moderately rewetted fen to a shallow lake - implications for net CO2 exchange

Science.gov (United States)

Koebsch, Franziska; Glatzel, Stephan; Hofmann, Joachim; Forbrich, Inke; Jurasinski, Gerald

2013-04-01

Extensive rewetting projects to re-establish the natural carbon (C) sequestration function of degraded peatlands are currently taking place in Europe and North-America. Year-round flooding provides a robust measure to prevent periods of drought that are associated with ongoing peat mineralization and to initiate the accumulation of new organic matter. Here, we present measurements of net carbon dioxide (CO2) exchange during the gradual conversion of a moderately rewetted fen to a shallow lake. When we started our measurements in 2009, mean growing season water level (MWGL) was 0 cm. In 2010 the site was flooded throughout the year with MWGL of 36 cm. Extraordinary strong rainfalls in July 2011 resulted in a further increase of MWGL to 56 cm. Measurements of net ecosystem exchange (NEE) were conducted during growing seasons (May-October) using the Eddy Covariance method. Information about vegetation vitality was deduced from the enhanced vegetation index (EVI) based on MODIS data. Ecosystem respiration (Reco) and gross ecosystem production (GEP) were high during vegetation period 2009 (1273.4 and -1572.1 g CO2-C m-2), but decreased by 61 and 46% respectively when the fen was flooded throughout 2010. Under water-logged conditions, heterotrophic respiration declines and gas exchange is limited. Moreover, flooding is a severe stress factor for plants and decreases autotrophic respiration and photosynthesis. However, in comparison to 2010, rates of Reco and GEP doubled during the beginning of growing season 2011, indicating plastic response strategies of wetland plants to flooding. Presumably, plants were not able to cope with the further increase of water levels to up to 120 cm in June/July 2011, resulting in another drop of GEP and Reco. The effects of plant vitality on GEP were confirmed by the remote sensed vegetation index. Throughout all three growing seasons, the fen was a distinct net CO2 sink (2009: -333.3±12.3, 2010: -294.1±8.4, -352.4±5.1 g CO2-C m-2
Produção de mudas de cultivares de alface utilizando duas espumas fenólicas em Altamira, Pará

Directory of Open Access Journals (Sweden)

Alzirio Lino Couto

2015-06-01

Full Text Available O sucesso de qualquer produção comercial de hortaliças inicia-se com a produção de mudas. A espuma fenólica tem se mostrado um interessante e prático substrato para este processo. A utilização deste produto depende de uma lavagem precedente para reduzir a acidez. Porém, já é possível encontrar à venda espumas fenólicas com pH próximo. Por isso o objetivo deste trabalho foi avaliar a produção de mudas de cultivares de alface utilizando duas espumas fenólicas em Altamira, Pará. O delineamento constou de um esquema fatorial 2 (substratos x 6 (cultivares. Os dois substratos foram as espumas fenólicas Green Up®, com pH 6.0 (± 0.5., e a outra com pH 2.6 (± 0.5. As cultivares avaliadas foram: Alcione, Amanda, Lucy Brown, Caipira, Multiblond 3 e Solaris. Foi avaliada porcentagem de germinação, índice de velocidade de emergência, altura da planta e número de folhas. Houve diferença significativa para altura da planta em função dos substratos testados. A porcentagem de germinação mais elevada foi obtida com a espuma fenólica sem tratamento. A cultivar Caipira foi a que teve o menor desempenho na germinação. Para todas as variáveis analisadas, a cultivar Lucy Brown apresentou os melhores resultados em ambos os substratos.
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.
Net ecosystem exchange in a sedge-sphagnum fen at the South of West Siberia, Russia

Science.gov (United States)

Dyukarev, Egor

2017-04-01

The model of net ecosystem exchange was used to study the influence of different environmental factors and to calculate daily and growing season carbon budget for minerotrophic fen at South of West Siberia, Russia. Minerotrophic sedge-sphagnum fen occupies the central part of the Bakcharskoe bog. The model uses air and soil temperature, incoming photosynthetically active radiation, and leaf area index as the explanatory factors for gross primary production, heterotrophic and autotrophic respiration. The model coefficients were calibrated using data collected by automated soil CO2 flux system with clear long-term chamber. The studied ecosystem is a sink of carbon according to modelling and observation results. This study was supported by Russian Foundation for Basic Researches (grant numbers 16-07-01205 and 16-45-700562.
A cellulose synthase-like protein is required for osmotic stress tolerance in Arabidopsis

KAUST Repository

Zhu, Jianhua

2010-04-16

Osmotic stress imposed by soil salinity and drought stress significantly affects plant growth and development, but osmotic stress sensing and tolerance mechanisms are not well understood. Forward genetic screens using a root-bending assay have previously identified salt overly sensitive (sos) mutants of Arabidopsis that fall into five loci, SOS1 to SOS5. These loci are required for the regulation of ion homeostasis or cell expansion under salt stress, but do not play a major role in plant tolerance to the osmotic stress component of soil salinity or drought. Here we report an additional sos mutant, sos6-1, which defines a locus essential for osmotic stress tolerance. sos6-1 plants are hypersensitive to salt stress and osmotic stress imposed by mannitol or polyethylene glycol in culture media or by water deficit in the soil. SOS6 encodes a cellulose synthase-like protein, AtCSLD5. Only modest differences in cell wall chemical composition could be detected, but we found that sos6-1 mutant plants accumulate high levels of reactive oxygen species (ROS) under osmotic stress and are hypersensitive to the oxidative stress reagent methyl viologen. The results suggest that SOS6/AtCSLD5 is not required for normal plant growth and development but has a critical role in osmotic stress tolerance and this function likely involves its regulation of ROS under stress. © 2010 Blackwell Publishing Ltd.
A first principles calculations of structural, electronic, magnetic and dynamical properties of mononitrides FeN and CoN

International Nuclear Information System (INIS)

Soni, Himadri R.; Mankad, Venu; Gupta, Sanjeev K.; Jha, Prafulla K.

2012-01-01

Highlights: ► We present spin dependent bandstructure, structural and magnetic moment of FeN/CoN. ► The PDC, PHDOS, spin effect on phonons suggests ZB is preferred at ambient pressure. ► Spin calculation offers an opportunity to understand the role of spin on phonons. - Abstract: Using first principles density functional theoretical calculations, the present paper reports a systematic nonspin and spin polarized total energy calculations of the lattice dynamical and a number of other properties such as band structure, structural and magnetic moment of two mononitrides FeN and CoN. The phonon dispersion curves and phonon density of states in the case of FeN and CoN have been determined for the first time and discussed. The structural and dynamical calculations suggest that the zinc blende structure is preferred at ambient pressure for both compounds. The rocksalt FeN has a nonzero magnetic moment while for FeN in zinc blende phase, it is either zero or very small. The zinc blende phase for both compounds is nonmagnetic. The spin calculation offers an intensive opportunity to understand the role of spin on the phonon properties of two mononitrides. Majority of the modes are sensitive to the effect of spin due to the modification of lattice constant. In this work we reveal that spin modifies the interionic interactions and local structure and leads to a flexible lattice which can be used for the functional materials design.
Determinação de compostos fenólicos em amostras comerciais de chás verde e preto - Camellia sinensis (L. Kuntze, Theaceae - DOI: 10.4025/actascihealthsci.v31i2.6233

Directory of Open Access Journals (Sweden)

Maria Eugênia Costa

2009-09-01

Full Text Available O chá de Camellia sinensis é rico em compostos fenólicos antioxidantes, responsáveis por efeitos benéficos à saúde humana. Neste trabalho, foram realizadas as determinações da concentração de fenóis totais e flavonoides de sete amostras comerciais dos chás preto e verde. Os teores de fenóis totais variaram de 4,80 a 26,60 mg de pirogalol g-1 de amostra e os de flavonoides, de 0,46 a 1,10 mg de quercetina g-1 de amostra. A análise anatômica do conteúdo das amostras permitiu caracterizar a autenticidade dos produtos, observando-se os marcadores anatômicos típicos da espécie (estômatos anomocíticos, tricomas tectores unicelulares, mesofilo dorsiventral, drusas de oxalato de cálcio e esclereides.
The Anti-Apoptotic Properties of APEX1 in the Endothelium Require the First 20 Amino Acids and Converge on Thioredoxin-1.

Science.gov (United States)

Dyballa-Rukes, Nadine; Jakobs, Philipp; Eckers, Anna; Ale-Agha, Niloofar; Serbulea, Vlad; Aufenvenne, Karin; Zschauer, Tim-Christian; Rabanter, Lothar L; Jakob, Sascha; von Ameln, Florian; Eckermann, Olaf; Leitinger, Norbert; Goy, Christine; Altschmied, Joachim; Haendeler, Judith

2017-04-20

The APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1) has a disordered N-terminus, a redox, and a DNA repair domain. APEX1 has anti-apoptotic properties, which have been linked to both domains depending on cell type and experimental conditions. As protection against apoptosis is a hallmark of vessel integrity, we wanted to elucidate whether APEX1 acts anti-apoptotic in primary human endothelial cells and, if so, what the underlying mechanisms are. APEX1 inhibits apoptosis in endothelial cells by reducing Cathepsin D (CatD) cleavage, potentially by binding to the unprocessed form. Diminished CatD activation results in increased Thioredoxin-1 protein levels leading to reduced Caspase 3 activation. Consequently, apoptosis rates are decreased. This depends on the first twenty amino acids in APEX1, because APEX1 (21-318) induces CatD activity, decreases Thioredoxin-1 protein levels, and, thus, increases Caspase 3 activity and apoptosis. Along the same lines, APEX1 (1-20) inhibits Caspase 3 cleavage and apoptosis. Furthermore, re-expression of Thioredoxin-1 via lentiviral transduction rescues endothelial cells from APEX1 (21-318)-induced apoptosis. In an in vivo model of restenosis, which is characterized by oxidative stress, endothelial activation, and smooth muscle cell proliferation, Thioredoxin-1 protein levels are reduced in the endothelium of the carotids. APEX1 acts anti-apoptotic in endothelial cells. This anti-apoptotic effect depends on the first 20 amino acids of APEX1. As proper function of the endothelium during life span is a hallmark for individual health span, a detailed characterization of the functions of the APEX1N-terminus is required to understand all its cellular properties. Antioxid. Redox Signal. 26, 616-629.
O fenómeno do Fansubbing em inglês: principais normas de tradução e legendagem

OpenAIRE

Sousa, Inês Fernandes e

2011-01-01

Tese de mestrado, Tradução, Universidade de Lisboa, Faculdade de Letras, 2012 Este trabalho enquadra-se no âmbito dos Estudos Descritivos de Tradução, mais concretamente, nos conceitos de norma e estratégia de tradução e teve como objectivo estudar algumas das principais estratégias e regularidades evidenciadas pelo fenómeno de “fansubbing”. Este fenómeno corresponde ao trabalho de equipas amadoras especializadas na tradução para legendagem predominantemente em inglês de desenh...
Metódica: Articulación sistema teórico, fenómeno y procesos en el objeto de estudio. Una perspectiva desde el fenómeno pobreza

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Yajaira Jiménez Zabala

2011-10-01

Full Text Available En este artículo discutimos la visión metódica que toma como apoyo el pivote entre lo cualitativo, lo cuantitativo y el orden de complejidad, en un enfoque plurimetódico, a través del cual se construyó la visión de la pobreza como objeto de estudio. Tal selección, está justificada en tanto se entiende la pobreza como un fenómeno social complejo, por las diversas aristas que le identifican. La combinación de los enfoques metodológicos permitió la aprehensión de los diferentes aspectos del fenómeno y de sus interrelaciones en las representaciones de los actores sociales vinculados con el estudio. Lo cualitativo, se entiende como la simbiosis de prácticas interpretativas, que no privilegia una metodología sobre otra. Lo cuantitativo estuvo referido al uso de indicadores derivados de los soportes referenciales del problema; el orden de complejidad, atendió a la manifestación de lo simple en tanto nodo tensional complejo, que requiere una mirada diferente para su comprensión e interpretación.
Generation of the SCN1A epilepsy mutation in hiPS cells using the TALEN technique

Science.gov (United States)

Chen, Wanjuan; Liu, Jingxin; Zhang, Longmei; Xu, Huijuan; Guo, Xiaogang; Deng, Sihao; Liu, Lipeng; Yu, Daiguan; Chen, Yonglong; Li, Zhiyuan

2014-06-01

Human induced pluripotent stem cells (iPSC) can be used to understand the pathological mechanisms of human disease. These cells are a promising source for cell-replacement therapy. However, such studies require genetically defined conditions. Such genetic manipulations can be performed using the novel Transcription Activator-Like Effector Nucleases (TALENs), which generate site-specific double-strand DNA breaks (DSBs) with high efficiency and precision. Combining the TALEN and iPSC methods, we developed two iPS cell lines by generating the point mutation A5768G in the SCN1A gene, which encodes the voltage-gated sodium channel Nav1.1 α subunit. The engineered iPSC maintained pluripotency and successfully differentiated into neurons with normal functional characteristics. The two cell lines differ exclusively at the epilepsy-susceptibility variant. The ability to robustly introduce disease-causing point mutations in normal hiPS cell lines can be used to generate a human cell model for studying epileptic mechanisms and for drug screening.
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
A decade of boreal rich fen greenhouse gas fluxes in response to natural and experimental water table variability

Science.gov (United States)

Olefeldt, David; Euskirchen, Eugénie S.; Harden, Jennifer W.; Kane, Evan S.; McGuire, A. David; Waldrop, Mark P.; Turetsky, Merritt R.

2017-01-01

Rich fens are common boreal ecosystems with distinct hydrology, biogeochemistry and ecology that influence their carbon (C) balance. We present growing season soil chamber methane emission (FCH4), ecosystem respiration (ER), net ecosystem exchange (NEE) and gross primary production (GPP) fluxes from a 9-years water table manipulation experiment in an Alaskan rich fen. The study included major flood and drought years, where wetting and drying treatments further modified the severity of droughts. Results support previous findings from peatlands that drought causes reduced magnitude of growing season FCH4, GPP and NEE, thus reducing or reversing their C sink function. Experimentally exacerbated droughts further reduced the capacity for the fen to act as a C sink by causing shifts in vegetation and thus reducing magnitude of maximum growing season GPP in subsequent flood years by ~15% compared to control plots. Conversely, water table position had only a weak influence on ER, but dominant contribution to ER switched from autotrophic respiration in wet years to heterotrophic in dry years. Droughts did not cause inter-annual lag effects on ER in this rich fen, as has been observed in several nutrient-poor peatlands. While ER was dependent on soil temperatures at 2 cm depth, FCH4 was linked to soil temperatures at 25 cm. Inter-annual variability of deep soil temperatures was in turn dependent on wetness rather than air temperature, and higher FCH4 in flooded years was thus equally due to increased methane production at depth and decreased methane oxidation near the surface. Short-term fluctuations in wetness caused significant lag effects on FCH4, but droughts caused no inter-annual lag effects on FCH4. Our results show that frequency and severity of droughts and floods can have characteristic effects on the exchange of greenhouse gases, and emphasize the need to project future hydrological regimes in rich fens.
Composição fenólica de ligninas dioxano determinadas pela reação oxidativa com o nitrobenzeno

Directory of Open Access Journals (Sweden)

Fukushima Romualdo Shigueo

2003-01-01

Full Text Available A correta quantificação da concentração de lignina em plantas forrageiras, pelo método espectrofotométrico, pressupõe a existência de um padrão de referência. Um padrão de referência deve ter composição fenólica semelhante à da lignina da parede celular. O objetivo deste trabalho foi verificar se ligninas extraídas com solução ácida de dioxano para serem utilizadas como padrão de referência, apresentariam variação na composição fenólica da mesma maneira que a lignina da parede celular. Amostras de parede celular de "bromegrass", milho e trevo-vermelho foram submetidas ao método para extração de ligninas com solução ácida de dioxano. A composição fenólica das ligninas foi analisada mediante oxidação alcalina pelo nitrobenzeno com posterior separação dos componentes monoméricos via HPLC. As ligninas extraídas confirmaram a existência de variação na composição fenólica da mesma maneira às ligninas intactas presentes na parede celular e de substancial presença de ácidos cinâmicos nas ligninas de gramíneas. No que diz respeito à composição fenólica, ligninas extraídas com solução ácida de dioxano podem ser consideradas potenciais padrões de referência paras as análises espectrofotométricas.
Zinc finger nuclease-mediated precision genome editing of an endogenous gene in hexaploid bread wheat (Triticum aestivum) using a DNA repair template.

Science.gov (United States)

Ran, Yidong; Patron, Nicola; Kay, Pippa; Wong, Debbie; Buchanan, Margaret; Cao, Ying-Ying; Sawbridge, Tim; Davies, John P; Mason, John; Webb, Steven R; Spangenberg, German; Ainley, William M; Walsh, Terence A; Hayden, Matthew J

2018-05-07

Sequence-specific nucleases have been used to engineer targeted genome modifications in various plants. While targeted gene knockouts resulting in loss of function have been reported with relatively high rates of success, targeted gene editing using an exogenously supplied DNA repair template and site-specific transgene integration has been more challenging. Here, we report the first application of zinc finger nuclease (ZFN)-mediated, nonhomologous end-joining (NHEJ)-directed editing of a native gene in allohexaploid bread wheat to introduce, via a supplied DNA repair template, a specific single amino acid change into the coding sequence of acetohydroxyacid synthase (AHAS) to confer resistance to imidazolinone herbicides. We recovered edited wheat plants having the targeted amino acid modification in one or more AHAS homoalleles via direct selection for resistance to imazamox, an AHAS-inhibiting imidazolinone herbicide. Using a cotransformation strategy based on chemical selection for an exogenous marker, we achieved a 1.2% recovery rate of edited plants having the desired amino acid change and a 2.9% recovery of plants with targeted mutations at the AHAS locus resulting in a loss-of-function gene knockout. The latter results demonstrate a broadly applicable approach to introduce targeted modifications into native genes for nonselectable traits. All ZFN-mediated changes were faithfully transmitted to the next generation. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
Interacting effects of sulphate pollution, sulphide toxicity and eutrophication on vegetation development in fens: A mesocosm experiment

Energy Technology Data Exchange (ETDEWEB)

Geurts, Jeroen J.M., E-mail: j.geurts@b-ware.e [Aquatic Ecology and Environmental Biology, Institute for Wetland and Water Research, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen (Netherlands); B-WARE Research Centre, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen (Netherlands); Sarneel, Judith M. [Landscape Ecology, Institute of Environmental Biology, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht (Netherlands); Willers, Bart J.C.; Roelofs, Jan G.M. [Aquatic Ecology and Environmental Biology, Institute for Wetland and Water Research, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen (Netherlands); Verhoeven, Jos T.A. [Landscape Ecology, Institute of Environmental Biology, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht (Netherlands); Lamers, Leon P.M. [Aquatic Ecology and Environmental Biology, Institute for Wetland and Water Research, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen (Netherlands)

2009-07-15

Both eutrophication and SO{sub 4} pollution can lead to higher availability of nutrients and potentially toxic compounds in wetlands. To unravel the interaction between the level of eutrophication and toxicity at species and community level, effects of SO{sub 4} were tested in nutrient-poor and nutrient-rich fen mesocosms. Biomass production of aquatic and semi-aquatic macrophytes and colonization of the water layer increased after fertilization, leading to dominance of highly competitive species. SO{sub 4} addition increased alkalinity and sulphide concentrations, leading to decomposition and additional eutrophication. SO{sub 4} pollution and concomitant sulphide production considerably reduced biomass production and colonization, but macrophytes were less vulnerable in fertilized conditions. The experiment shows that competition between species, vegetation succession and terrestrialization are not only influenced by nutrient availability, but also by toxicity, which strongly interacts with the level of eutrophication. This implies that previously neutralized toxicity effects in eutrophied fens may appear after nutrient reduction measures have been taken. - Interspecific competition, vegetation succession and terrestrialization in fens depend on the interacting effects of SO{sub 4} pollution, sulphide toxicity and nutrient availability.
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.
a fenómenos epidemiológicos

Directory of Open Access Journals (Sweden)

Carlos Arturo Castro Castro

2005-01-01

Full Text Available En este artículo se presentan avances del proyecto de investigación modelación y simulación computacional usando sistemas de información geográfica (SIG con dinámicas de sistemas aplicados a fenómenos epidemiológicos con el cual se han abordado los principales modelos matemáticos usados en epidemiología (susceptible- infectado-recuperado: SIR, susceptible-infectado-recuperado-susceptible: SIRS, susceptible-infectado-susceptible: SIS, para su integración, espacialización y simulación usando diferentes técnicas computacionales con el fin de modelar factores críticos en la propagación de epidemias. Para correr el modelo, se tomaron los datos de una epidemia de asma en el distrito de Manhattan de Nueva York, del tutotial de ARCGIS® versión 8.3.
Human RECQL5beta stimulates flap endonuclease 1

DEFF Research Database (Denmark)

Speina, Elzbieta; Dawut, Lale; Hedayati, Mohammad

2010-01-01

devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5beta, with the human flap endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5beta...... dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests...
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.
Fenómenos que Organizan el Límite (Phenomena that Organize Limits

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María Teresa Sánchez

2007-03-01

Full Text Available En este artículo se pone de manifiesto la presencia de los fenómenos de aproximación organizados por una definición de límite en el caso de las sucesiones de números reales y de las funciones reales de una variable real. La exposición incluye la caracterización de tales fenómenos, una descripción del análisis comparativo desarrollado en base a ellos entre dos definiciones formales de límite de sucesión y función, y una síntesis del estudio llevado a cabo sobre una muestra intencional de libros de texto de matemáticas.In this paper we show the presence of the phenomena of approximation organized by a definition of limit in the cases of the successions of real numbers and the real functions of one real variable. The exposition includes the characterization of such phenomena, a description of the comparative analysis, developed using them, between two formal definitions of limit of succession and function, and a synthesis of the study carried out on an intentional sample of mathematics textbooks.
TRF2 Recruits RTEL1 to Telomeres in S Phase to Promote T-Loop Unwinding

OpenAIRE

Sarek, Grzegorz; Vannier, Jean-Baptiste; Panier, Stephanie; Petrini, John?H.J.; Boulton, Simon?J.

2015-01-01

Summary The helicase RTEL1 promotes t-loop unwinding and suppresses telomere fragility to maintain the integrity of vertebrate telomeres. An interaction between RTEL1 and PCNA is important to prevent telomere fragility, but how RTEL1 engages with the telomere to promote t-loop unwinding is unclear. Here, we establish that the shelterin protein TRF2 recruits RTEL1 to telomeres in S phase, which is required to?prevent catastrophic t-loop processing by structure-specific nucleases. We show that ...
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.
Cathepsins are required for Toll-like receptor 9 responses

International Nuclear Information System (INIS)

Matsumoto, Fumi; Saitoh, Shin-ichiroh; Fukui, Ryutaroh; Kobayashi, Toshihiko; Tanimura, Natsuko; Konno, Kazunori; Kusumoto, Yutaka; Akashi-Takamura, Sachiko; Miyake, Kensuke

2008-01-01

Toll-like receptors (TLR) recognize a variety of microbial products and activate defense responses. Pathogen sensing by TLR2/4 requires accessory molecules, whereas little is known about a molecule required for DNA recognition by TLR9. After endocytosis of microbes, microbial DNA is exposed and recognized by TLR9 in lysosomes. We here show that cathepsins, lysosomal cysteine proteases, are required for TLR9 responses. A cell line Ba/F3 was found to be defective in TLR9 responses despite enforced TLR9 expression. Functional cloning with Ba/F3 identified cathepsin B/L as a molecule required for TLR9 responses. The protease activity was essential for the complementing effect. TLR9 responses were also conferred by cathepsin S or F, but not by cathepsin H. TLR9-dependent B cell proliferation and CD86 upregulation were apparently downregulated by cathepsin B/L inhibitors. Cathepsin B inhibitor downregulated interaction of CpG-B with TLR9 in 293T cells. These results suggest roles for cathepsins in DNA recognition by TLR9
Research of Ancient Architectures in Jin-Fen Area Based on GIS&BIM Technology

Science.gov (United States)

Jia, Jing; Zheng, Qiuhong; Gao, Huiying; Sun, Hai

2017-05-01

The number of well-preserved ancient buildings located in Shanxi Province, enjoying the absolute maximum proportion of ancient architectures in China, is about 18418, among which, 9053 buildings have the structural style of wood frame. The value of the application of BIM (Building Information Modeling) and GIS (Geographic Information System) is gradually probed and testified in the corresponding fields of ancient architecture’s spatial distribution information management, routine maintenance and special conservation & restoration, the evaluation and simulation of related disasters, such as earthquake. The research objects are ancient architectures in JIN-FEN area, which were first investigated by Sicheng LIANG and recorded in his work of “Chinese ancient architectures survey report”. The research objects, i.e. the ancient architectures in Jin-Fen area include those in Sicheng LIANG’s investigation, and further adjustments were made through authors’ on-site investigation and literature searching & collection. During this research process, the spatial distributing Geodatabase of research objects is established utilizing GIS. The BIM components library for ancient buildings is formed combining on-site investigation data and precedent classic works, such as “Yingzao Fashi”, a treatise on architectural methods in Song Dynasty, “Yongle Encyclopedia” and “Gongcheng Zuofa Zeli”, case collections of engineering practice, by the Ministry of Construction of Qing Dynasty. A building of Guangsheng temple in Hongtong county is selected as an example to elaborate the BIM model construction process based on the BIM components library for ancient buildings. Based on the foregoing work results of spatial distribution data, attribute data of features, 3D graphic information and parametric building information model, the information management system for ancient architectures in Jin-Fen Area, utilizing GIS&BIM technology, could be constructed to support the
Response of anaerobic carbon cycling to water table manipulation in an Alaskan rich fen

Science.gov (United States)

Kane, E.S.; Chivers, M.R.; Turetsky, M.R.; Treat, C.C.; Petersen, D.G.; Waldrop, M.; Harden, J.W.; McGuire, A.D.

2013-01-01

To test the effects of altered hydrology on organic soil decomposition, we investigated CO2 and CH4 production potential of rich-fen peat (mean surface pH = 6.3) collected from a field water table manipulation experiment including control, raised and lowered water table treatments. Mean anaerobic CO2 production potential at 10 cm depth (14.1 ± 0.9 μmol C g−1 d−1) was as high as aerobic CO2 production potential (10.6 ± 1.5 μmol C g−1 d−1), while CH4 production was low (mean of 7.8 ± 1.5 nmol C g−1 d−1). Denitrification enzyme activity indicated a very high denitrification potential (197 ± 23 μg N g−1 d−1), but net NO-3 reduction suggested this was a relatively minor pathway for anaerobic CO2 production. Abundances of denitrifier genes (nirK and nosZ) did not change across water table treatments. SO2-4 reduction also did not appear to be an important pathway for anaerobic CO2 production. The net accumulation of acetate and formate as decomposition end products in the raised water table treatment suggested that fermentation was a significant pathway for carbon mineralization, even in the presence of NO-3. Dissolved organic carbon (DOC) concentrations were the strongest predictors of potential anaerobic and aerobic CO2 production. Across all water table treatments, the CO2:CH4 ratio increased with initial DOC leachate concentrations. While the field water table treatment did not have a significant effect on mean CO2 or CH4 production potential, the CO2:CH4 ratio was highest in shallow peat incubations from the drained treatment. These data suggest that with continued drying or with a more variable water table, anaerobic CO2 production may be favored over CH4 production in this rich fen. Future research examining the potential for dissolved organic substances to facilitate anaerobic respiration, or alternative redox processes that limit the effectiveness of organic acids as substrates in anaerobic metabolism, would help explain additional
Environmental and Physiographic Controls on Inter-Growing Season Variability of Carbon Dioxide and Water Vapour Fluxes in a Minerotrophic Fen

Science.gov (United States)

van der Kamp, G.; Sonnentag, O.; Chen, J. M.; Barr, A.; Hedstrom, N.; Granger, R.

2008-12-01

The interaction of fens with groundwater is spatially and temporally highly variable in response to meteorological conditions, resulting in frequent changes of groundwater fluxes in both vertical and lateral directions (flow reversals) across the mineral soil-peat boundary. However, despite the importance of the topographic and hydrogeological setting of fens, no study has been reported in the literature that explores a fen's atmospheric CO2 and energy flux densities under contrasting meteorological conditions in response to its physiographic setting. In our contribution we report four years of growing season eddy covariance and supporting measurements from the Canada Fluxnet-BERMS fen (formerly BOREAS southern peatland) in Saskatchewan, Canada. We first analyze hydrological data along two piezometer transects across the mineral soil-peat boundary with the objective of assessing changes in water table configuration and thus hydraulic gradients, indicating flow reversals, in response to dry and wet meteorological conditions. Next we quantify and compare growing season totals and diurnal and daily variations in evapotranspiration (ET) and net ecosystem exchange (NEE) and its component fluxes gross ecosystem productivity (GPP) and terrestrial ecosystem respiration (TER) to identify their controls with a major focus on water table depth. While ET growing season totals were similar (~ 310 mm) under dry and wet meteorological conditions, the CO2 sink- source strength of Sandhill fen varied substantially from carbon neutral (NEE = -2 [+-7] g C m-2 per growing season) under dry meteorological condition (2003) to a moderate CO2- sink with NEE ranging between 157 [+- 10] and 190 [+- 11] g C m-2 per growing season under wet meteorological conditions (2004, 2005, and 2006). Using a process-oriented ecosystem model, BEPS-TerrainLab, we investigate how different canopy components at Sandhill contribute to total ET and GPP, and thus water use efficiency, under dry and wet
Violência como fenômeno intrínseco à cultura política brasileira

Directory of Open Access Journals (Sweden)

Antonio M. Soares

2016-06-01

Full Text Available Este artigo problematiza o conceito de violência e suas mutações através dos tempos. Como fenômeno cultural, a violência é construída na sociedade e se processa de forma a romper com os próprios códigos de ordens produzidos por ela. No caso do Brasil a violência se mostra intrínseca ao processo de formação da sociedade brasileira, estando presente nas lógicas autoritárias e na imposição cultural desde os tempos da colonização. A violência emerge como uma agressão ao indivíduo e ao coletivo, ela aniquila o sentido da política e desabilita o mundo comum. As interpretações presentes neste trabalho buscam aprofundar as análises sobre a violência enquanto fenômeno social e cultural, redimensionando os efeitos que ela produz para a condição humana e suas associações com as dimensões da civilidade, incivilidade e descivilidade. Neste contexto, destacaremos como o fenômeno da violência se afirmou como uma dimensão estrutural e cultural da formação da sociedade brasileira, como ele se revela na contemporaneidade, nos jogos de poder, nas relações de força e no movimento de fronteiras tênues entre o legal e o ilegal.
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...
Análisis de la influencia del fenómeno del ciberterrorismo en las dinámicas de seguridad de la Unión Europea

OpenAIRE

Torres Pedraza, Julián Alejandro

2014-01-01

El fenómeno del ciberterrorismo se constituye como una nueva amenaza a la seguridad internacional, este fenómeno es el resultado de distintos procesos en el sistema internacional como el de la globalización. La definición del concepto se establece por la convergencia entre el ciberespacio y el terrorismo. El objetivo de esta investigación es explicar las distintas dimensiones del ciberterrorismo, métodos de ataque, acciones pasadas, propósito del fenómeno, situación actual y vulnerabilidades....
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

El fenómeno del acoso laboral entre los trabajadores de la universidad O fenômeno mobbing entre os trabalhadores da universidade The phenomenon of mobbing among university employees

Directory of Open Access Journals (Sweden)

Fernando Justicia Justicia

2007-12-01

Full Text Available El objetivo del artículo es realizar una descripción del fenómemo mobbing -acoso laboral- en el contexto de la universidad. Los trabajos realizadossobre este problema no son muy prolíficos lo que puede explicar la gran dificultad para caracterizar el fenómeno en el ámbito universitario. En el presente trabajo presentamos los niveles de incidencia del acoso laboral entre la población universitaria, los tipos de acoso más frecuentes y los efectos producidos en las víctimas, con la finalidad de caracterizar y definir el problema. Al mismo tiempo, intentamos conocer la influencia de determinadas variables como la edad, género y tiempo de victimización. En este sentido, y de acuerdo con otros estudios realizados, encontramos diferencias estadísticas en función del género de las víctimas. Del mismo modo, los resultados apuntan que la frecuencia y duración del acoso laboral afectan negativamente a las víctimas provocando un empeoramiento de los efectos derivados del acoso.O objetivo deste artigo é realizar uma descrição do fenômeno mobbing -tormento e persecução no lugar do trabalho- no contexto da universidade. Há poucos trabalhos realizados sob este problema nos contextos de educação superior o que pode explicar a grande dificuldade para caracterizar o fenômeno no âmbito universitário. Neste artigo apresentamos os níveis de incidência entre a população universitária, os tipos de mobbing mais freqüentes e os efeitos produzidos nas vítimas com a finalidade de caracterizar e definir o problema. Ao mesmo tempo, tentamos conhecer a influência de determinadas variáveis tais como a idade, gênero ou o tempo de exposição ao fenômeno. Neste sentido, e em concordância com outros estudos realizados, encontramos diferenças estatísticas em função do gênero das vítimas. De igual forma, os dados apontam que a freqüência e duração da situação de persecução no trabalho afetam negativamente as vítimas provocando
SWI/SNF-like chromatin remodeling factor Fun30 supports point centromere function in S. cerevisiae.

Directory of Open Access Journals (Sweden)

Mickaël Durand-Dubief

2012-09-01

Full Text Available Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture.
Rehabilitation of acidified and eutrophied fens in The Netherlands : Effects of hydrologic manipulation and liming

NARCIS (Netherlands)

Beltman, B.; Broek, T. van den; Barendregt, A.; Bootsma, M.C.; Grootjans, A.P.

Mosaic fen communities with many rare plant species were outcompeted by fast-growing bryophyte stands (Sphagnum flexuosum/Sphagnum fallax and Polytrichum commune). Eutrophication and acidification were the main causes. Management experiments were carried out to set back this succession by restoring
Design, construction, and analysis of specific zinc finger nucleases for microphthalmia - associate transcription factor

Directory of Open Access Journals (Sweden)

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.
Suppression of HBV replication by the expression of nickase- and nuclease dead-Cas9.

Science.gov (United States)

Kurihara, Takeshi; Fukuhara, Takasuke; Ono, Chikako; Yamamoto, Satomi; Uemura, Kentaro; Okamoto, Toru; Sugiyama, Masaya; Motooka, Daisuke; Nakamura, Shota; Ikawa, Masato; Mizokami, Masashi; Maehara, Yoshihiko; Matsuura, Yoshiharu

2017-07-21

Complete removal of hepatitis B virus (HBV) DNA from nuclei is difficult by the current therapies. Recent reports have shown that a novel genome-editing tool using Cas9 with a single-guide RNA (sgRNA) system can cleave the HBV genome in vitro and in vivo. However, induction of a double-strand break (DSB) on the targeted genome by Cas9 risks undesirable off-target cleavage on the host genome. Nickase-Cas9 cleaves a single strand of DNA, and thereby two sgRNAs are required for inducing DSBs. To avoid Cas9-induced off-target mutagenesis, we examined the effects of the expressions of nickase-Cas9 and nuclease dead Cas9 (d-Cas9) with sgRNAs on HBV replication. The expression of nickase-Cas9 with a pair of sgRNAs cleaved the target HBV genome and suppressed the viral-protein expression and HBV replication in vitro. Moreover, nickase-Cas9 with the sgRNA pair cleaved the targeted HBV genome in mouse liver. Interestingly, d-Cas9 expression with the sgRNAs also suppressed HBV replication in vitro without cleaving the HBV genome. These results suggest the possible use of nickase-Cas9 and d-Cas9 with a pair of sgRNAs for eliminating HBV DNA from the livers of chronic hepatitis B patients with low risk of undesirable off-target mutation on the host genome.
MoDUO1, a Duo1-like gene, is required for full virulence of the rice blast fungus Magnaporthe oryzae.

Science.gov (United States)

Peng, Haowen; Feng, Youjun; Zhu, Xiaohui; Lan, Xiuwan; Tang, Mei; Wang, Jinzi; Dong, Haitao; Chen, Baoshan

2011-12-01

Duo1, a major component of the Dam1 complex which has been found in two species of yeast (the budding yeast Saccharomyces cerevisae and the fission yeast Schizosaccharomyces pombe), is involved in mitosis-related chromosome segregation, while its relevance to pathogenicity in filamentous fungi remains unclear. This report elucidated this very fact in the case of the rice blast fungus Magnaporthe oryzae. A gene designated MoDUO1 that encodes a Duo1-like homolog (MoDuo1) was discovered in the M. oryzae genome. Two types of MoDUO1 mutants were obtained using genetic approaches of Agrobacterium-mediated gene disruption and homologous recombination. Both disruption and deletion of MoDUO1 can exert profound effects on the formation pattern of conidiophores and conidial morphology, such as abnormal nucleic numbers in conidia and delayed extension of infectious hyphae. Intriguingly, plant infection assays demonstrated that inactivation of MoDUO1 significantly attenuates the virulence in its natural host rice leaves, and functional complementation can restore it. Subcellular localization assays showed that MoDuo1 is mainly distributed in the cytosol of fungal cells. Proteomics-based investigation revealed that the expression of four mitosis-related proteins is shut down in the MoDUO1 mutant, suggesting that MoDuo1 may have a function in mitosis. In light of the fact that Duo1 orthologs are widespread in plant and human fungal pathogens, our finding may represent a common mechanism underlying fungal virulence. To the best of our knowledge, this is the first example of linking a Duo1-like homolog to the pathogenesis of a pathogenic fungus, which might provide clues to additional studies on the role of Dam1 complex in M. oryzae and its interaction with rice.
Full GHG balance of a drained fen peatland cropped to spring barley and reed canary grass using comparative assessment of CO2 fluxes.

Science.gov (United States)

Karki, Sandhya; Elsgaard, Lars; Kandel, Tanka P; Lærke, Poul Erik

2015-03-01

Empirical greenhouse gas (GHG) flux estimates from diverse peatlands are required in order to derive emission factors for managed peatlands. This study on a drained fen peatland quantified the annual GHG balance (Carbon dioxide (CO2), nitrous oxide (N2O), methane (CH4), and C exported in crop yield) from spring barley (SB) and reed canary grass (RCG) using static opaque chambers for GHG flux measurements and biomass yield for indirectly estimating gross primary production (GPP). Estimates of ecosystem respiration (ER) and GPP were compared with more advanced but costly and labor-intensive dynamic chamber studies. Annual GHG balance for the two cropping systems was 4.0 ± 0.7 and 8.1 ± 0.2 Mg CO2-Ceq ha(-1) from SB and RCG, respectively (mean ± standard error, n = 3). Annual CH4 emissions were negligible (peatland cropped to SB and RCG and presented a valid alternative to estimating the full GHG balance by dynamic chambers.
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
Response of anaerobic carbon cycling to water table manipulation in an Alaskan rich fen

Science.gov (United States)

E.S. Kane; M.R. Chivers; M.S. Turetsky; C.C. Treat; D.G. Petersen; M. Waldrop; J.W. Harden; A.D. McGuire

2013-01-01

To test the effects of altered hydrology on organic soil decomposition, we investigated CO2 and CH4 production potential of rich-fen peat (mean surface pH = 6.3) collected from a field water table manipulation experiment including control, raised and lowered water table treatments. Mean anaerobic CO2...
Partitioning of habitat effects casts light on the decline of the fen orchid, Liparis loeselii

DEFF Research Database (Denmark)

Andersen, Dagmar Kappel; Ejrnæs, Rasmus; Minter, Martine Olesen

2015-01-01

Liparis loeselii is a rare and declining orchid species restricted to rich fens in the northern hemisphere. Suggested reasons for the decline are habitat destruction, eutrophication, altered hydrology and scrub encroachment after termination of traditional management such as grazing and hay making...
Biodegradación de compuestos fenólicos del alpechín con Aspergillus terreus

Directory of Open Access Journals (Sweden)

García Paeja, María P.

1992-04-01

Full Text Available Olive mill wastewater degradation by Aspergillus terreus and under aerobic conditions at 28°C, was measured by the parameter of phenol content. We have explored the effect of different concentrations of olive mill wastewater upon the activity of Aspergillus terreus. Through HPLC, 10 phenol compounds (90% of the total phenolic content of the olive mill wastewater were identified.Se estudia la degradación de alpechines con Aspergillus terreus en condiciones de aerobiosis y temperatura de 28°C, utilizando como parámetro el contenido fenólico. Se analiza el efecto de la concentración de alpechín con Aspergillus terreus utilizando el mismo parámetro. Se han identificado por cromatografía líquida de alta eficacia (CLAE 10 compuestos fenólicos que suponen el 90% del total del alpechín.
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.
Phase transformations in interstitial Fe-N alloys

Energy Technology Data Exchange (ETDEWEB)

Liapina, T.

2005-04-15

Nitriding is a prominent thermochemical heat-treatment procedure leading to various types of surface property improvements of the treated iron and steel. Many questions regarding even very basic properties of these compound layers and the relevant nitride phases are still open. Some of these open questions related with the behaviour of iron nitrides and, in particular, of iron nitride compound layers occurring below the usual process temperatures are addressed to in this thesis, as relevant e.g. for the cooling procedure after nitriding. The most important iron nitrides occurring in iron-nitride compound layers are the {gamma}'- and {epsilon}-phases in the Fe-N system. It is shown that for relatively low nitrogen contents of epsilon-iron nitrides (around Fe{sub 3}N) the cooling rate upon going down from an elevated annealing temperature to room temperature has a significant effect on the lattice parameters. X-ray and neutron diffraction analysis revealed that the lattice parameter values observed after fast cooling are affected by the higher degree of nitrogen disorder at elevated temperature, thus changing the c/a ratio. New relations between the lattice parameters of {epsilon}-iron nitrides and the nitrogen content are suggested for different types of cooling. The investigation by TEM of the decomposition upon annealing (633 K, 673 K) of initially homogeneous {epsilon}-Fe{sub 3}N powders revealed that the {gamma}'-formation occurs in only a few powder particles in a grain-like form. Moreover, diffraction line-profile analysis revealed N transport occurring from particle to particle, leading to inhomogeneities of N content in the epsilon-phase. It was shown that {gamma}'-iron nitride formation can occur by backwards growth of the existing {gamma}'-sublayer at the cost of the {epsilon}-sublayer increasing N concentration in the {epsilon}-layer. Another process, which may additionally occur in the compound layer upon annealing, is diffusion of N
Effects of Reduced and Oxidised Nitrogen on Rich-Fen Mosses: a 4-Year Field Experiment

NARCIS (Netherlands)

Paulissen, Maurice P.C.P.; Bobbink, Roland; Robat, Sandra A.; Verhoeven, Jos T.A.

2016-01-01

Dutch fens, subjected to high nitrogen (N) deposition levels with reduced N (NHy) highly dominating over oxidised N (NOx), have since the second half of the past century seen a significant decline of Scorpidium and other characteristic brown moss species, while several Sphagnum species have
Voces actuales en el fenómeno de pasantes de drogas ilícitas en Colombia

Directory of Open Access Journals (Sweden)

Galia J. Benítez

2017-12-01

Full Text Available Este documento se deriva del análisis realizado a las narrativas de los pasantes de drogas ilícitas denominados popularmente como “mulas”; de manera especial, resalta su mirada localizada sobre el fenómeno del tráfico ilegal de drogas en Colombia. El escrito tiene como objetivo: primero, caracterizar la multiplicidad de conceptos que se utilizan para describir este fenómeno y proponer una definición que abarque diferentes acepciones, y segundo, presentar categorías conceptuales que se desprenden de las narrativas de los pasantes de drogas. Como metodología se utilizó el análisis de contenido, para describir, sistematizar y analizar los testimonios voluntarios de 42 personas imputadas y condenadas como pasantes de drogas ilícitas en Colombia. Esta investigación argumenta que uno de los elementos para entender el fenómeno de los pasantes de drogas ilícitas y su experiencia es su condición de vulnerabilidad. Por un lado, los pasantes ocupan un lugar periférico en las organizaciones narcotraficantes; por otro, son altamente vulnerables al sistema socio-técnico establecido por el Estado para prevenir el transporte de drogas ilícitas. Este estudio muestra que las narrativas de los pasantes de drogas se pueden categorizar en cuatro dicotomías, que sirven como elementos retóricos para la ruptura del binario legalidad/ilegalidad.
1 CFR 10.1 - Publication required.

Science.gov (United States)

2010-01-01

... 1 General Provisions 1 2010-01-01 2010-01-01 false Publication required. 10.1 Section 10.1 General Provisions ADMINISTRATIVE COMMITTEE OF THE FEDERAL REGISTER SPECIAL EDITIONS OF THE FEDERAL REGISTER PRESIDENTIAL PAPERS Regular Publication § 10.1 Publication required. The Director publishes a special edition...
Research of Ancient Architectures in Jin-Fen Area Based on GIS and BIM Technology

International Nuclear Information System (INIS)

Jia, Jing; Zheng, Qiuhong; Gao, Huiying; Sun, Hai

2017-01-01

The number of well-preserved ancient buildings located in Shanxi Province, enjoying the absolute maximum proportion of ancient architectures in China, is about 18418, among which, 9053 buildings have the structural style of wood frame. The value of the application of BIM (Building Information Modeling) and GIS (Geographic Information System) is gradually probed and testified in the corresponding fields of ancient architecture’s spatial distribution information management, routine maintenance and special conservation and restoration, the evaluation and simulation of related disasters, such as earthquake. The research objects are ancient architectures in JIN-FEN area, which were first investigated by Sicheng LIANG and recorded in his work of “Chinese ancient architectures survey report”. The research objects, i.e. the ancient architectures in Jin-Fen area include those in Sicheng LIANG’s investigation, and further adjustments were made through authors’ on-site investigation and literature searching and collection. During this research process, the spatial distributing Geodatabase of research objects is established utilizing GIS. The BIM components library for ancient buildings is formed combining on-site investigation data and precedent classic works, such as “Yingzao Fashi”, a treatise on architectural methods in Song Dynasty, “Yongle Encyclopedia” and “Gongcheng Zuofa Zeli”, case collections of engineering practice, by the Ministry of Construction of Qing Dynasty. A building of Guangsheng temple in Hongtong county is selected as an example to elaborate the BIM model construction process based on the BIM components library for ancient buildings. Based on the foregoing work results of spatial distribution data, attribute data of features, 3D graphic information and parametric building information model, the information management system for ancient architectures in Jin-Fen Area, utilizing GIS and BIM technology, could be constructed to support
Avaliação da influência de substâncias fenólicas e carotenoides na anomalia do epicarpo da goiaba, "anelamento"

Directory of Open Access Journals (Sweden)

Taiji Watanabe

2011-03-01

Full Text Available A anomalia do epicarpo da goiaba, comumente relatada por agricultores e técnicos como o "anelamento juvenil da goiaba", tem causado preocupação devido à desinformação sobre o assunto. O objetivo deste estudo foi analisar quimicamente as concentrações de substâncias fenólicas e carotenoides na região do epicarpo de goiabas afetadas pelo "anelamento", visando a caracterizar essa anomalia previamente relatada. Foram analisadas substâncias fenólicas (taninos, flavonas/flavonóis, antocianinas e fenóis totais e carotenoides em epicarpos de frutos verdes e maduros de goiabeiras cv. Paluma, com e sem anomalia. O delineamento experimental adotado foi o inteiramente casualizado, sendo estabelecidos seis tratamentos com o epicarpo dos frutos maduro sem anomalia na região inferior (FMSI; frutos maduros sem injuria na região superior (FMSS; frutos verdes sem anomalia na região inferior (FVSI; frutos verdes sem anomalia na região superior (FVSS; frutos verdes com anomalia na região inferior (FVCI; frutos verdes com anomalia na região superior (FVCS. Dentre as substâncias analisadas, os carotenoides, os taninos e os fenóis totais mostram indicativos para a caracterização do anelamento. Tanto substâncias fenólicas quanto carotenoides apresentam propriedades antioxidantes e, dessa forma, poderiam estar relacionadas à defesa antioxidante causada por um fator de estresse ainda desconhecido, que promove o "anelamento" característico apresentado pelas goiabas.
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.
Methane Exchange in a Coastal Fen in the First Year after Flooding - A Systems Shift

Science.gov (United States)

Hahn, Juliane; Köhler, Stefan; Glatzel, Stephan; Jurasinski, Gerald

2015-01-01

Background Peatland restoration can have several objectives, for example re-establishing the natural habitat, supporting unique biodiversity attributes or re-initiating key biogeochemical processes, which can ultimately lead to a reduction in greenhouse gas (GHG) emissions. Every restoration measure, however, is itself a disturbance to the ecosystem. Methods Here, we examine an ecosystem shift in a coastal fen at the southern Baltic Sea which was rewetted by flooding. The analyses are based on one year of bi-weekly closed chamber measurements of methane fluxes gathered at spots located in different vegetation stands. During measurement campaigns, we recorded data on water levels, peat temperatures, and chemical properties of peat water. In addition we analyzed the first 20 cm of peat before and after flooding for dry bulk density (DBD), content of organic matter and total amounts of carbon (C), nitrogen (N), sulfur (S), and other nutrients. Results Rewetting turned the site from a summer dry fen into a shallow lake with water levels up to 0.60 m. We observed a substantial die-back of vegetation, especially in stands of sedges (Carex acutiformis Ehrh). Concentrations of total organic carbon and nitrogen in the peat water, as well as dry bulk density and concentrations of C, N and S in the peat increased. In the first year after rewetting, the average annual exchange of methane amounted to 0.26 ± 0.06 kg m-2. This is equivalent to a 190-times increase in methane compared to pre-flooding conditions. Highest methane fluxes occurred in sedge stands which suffered from the heaviest die-back. None of the recorded environmental variables showed consistent relationships with the amounts of methane exchanged. Conclusions Our results suggest that rewetting projects should be monitored not only with regard to vegetation development but also with respect to biogeochemical conditions. Further, high methane emissions that likely occur directly after rewetting by flooding should

Single-Step Generation of Conditional Knockout Mouse Embryonic Stem Cells

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Matyas Flemr

2015-07-01

Full Text Available Induction of double-strand DNA breaks (DSBs by engineered nucleases, such as CRISPR/Cas9 or transcription activator-like effector nucleases (TALENs, stimulates knockin of exogenous DNA fragments via homologous recombination (HR. However, the knockin efficiencies reported so far have not allowed more complex in vitro genome modifications such as, for instance, simultaneous integration of a DNA fragment at two distinct genomic sites. We developed a reporter system to enrich for cells with engineered nuclease-assisted HR events. Using this system in mouse embryonic stem cells (mESCs, we achieve single-step biallelic and seamless integration of two loxP sites for Cre recombinase-mediated inducible gene knockout, as well as biallelic endogenous gene tagging with high efficiency. Our approach reduces the time and resources required for conditional knockout mESC generation dramatically.
1 CFR 9.1 - Publication required.

Science.gov (United States)

2010-01-01

... 1 General Provisions 1 2010-01-01 2010-01-01 false Publication required. 9.1 Section 9.1 General Provisions ADMINISTRATIVE COMMITTEE OF THE FEDERAL REGISTER SPECIAL EDITIONS OF THE FEDERAL REGISTER THE UNITED STATES GOVERNMENT MANUAL § 9.1 Publication required. The Director of the Federal Register shall...
Avaliação do teor de antioxidantes, flavonoides e compostos fenólicos em preparações condimentares

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G.M. MENDES

2015-06-01

Full Text Available RESUMO:Plantas condimentares são amplamente utilizadas na culinária como alimento funcional. Pacientes hipertensos, que retiram o sal de cozinha (NaCl de sua alimentação, relatam que os alimentos "perdem" o sabor. Visando alternativas para substituir o sal e melhorar a palatabilidade das preparações alimentares de pacientes hipertensos, o objetivo deste estudo foi desenvolver temperos à base de plantas medicinais e analisar os teores de compostos fenólicos, flavonoides e atividade antioxidante destes. Plantas (coentro, salsa, orégano, alecrim, manjericão, alho, cebola cultivadas organicamente em Viçosa (MG foram processadas e os temperos foram desenvolvidos a partir de combinação destas espécies, de acordo com teste aceitabilidade sensorial afetivo por meio de escala hedônica. Foram definidos três temperos, dos quais foram feitos extratos metanólicos por remaceração até esgotamento. Nas dosagens utilizou-se DPPH na avaliação da atividade antioxidante; reagente de Folin-Ciocalteu para compostos fenólicos e vanilina clorídrica para flavonoides. As análises foram feitas com quatro repetições e os resultados submetidos à análise de variância e teste de média a 5% de significância. Na aceitação subjetiva dos temperos a nota média foi 7,07±1,09 e 8,0±0,93 na ação afetiva como alimento. O teor mais elevado de compostos fenólicos foi no tempero 2 (alho, salsa e manjericão (55,04±4,307 mg/mL, a maior ação antioxidante foi no tempero 1 (alho, cebola, coentro e orégano nos tempos 0 (48,72% e 30 minutos (78,37%. Os teores de flavonoides foram semelhantes nos 3 temperos. Todos os temperos tiveram boa aceitação por pacientes hipertensos e mostraram-se promissores na substituição dos condimentos convencionais.
Multiple and variable NHEJ-like genes are involved in resistance to DNA damage in Streptomyces ambofaciens

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Grégory Hoff

2016-11-01

Full Text Available Non homologous end-joining (NHEJ is a double strand break (DSB repair pathway which does not require any homologous template and can ligate two DNA ends together. The basic bacterial NHEJ machinery involves two partners: the Ku protein, a DNA end binding protein for DSB recognition and the multifunctional LigD protein composed a ligase, a nuclease and a polymerase domain, for end processing and ligation of the broken ends. In silico analyses performed in the 38 sequenced genomes of Streptomyces species revealed the existence of a large panel of NHEJ-like genes. Indeed, ku genes or ligD domain homologues are scattered throughout the genome in multiple copies and can be distinguished in two categories: the core NHEJ gene set constituted of conserved loci and the variable NHEJ gene set constituted of NHEJ-like genes present in only a part of the species. In Streptomyces ambofaciens ATCC 23877, not only the deletion of core genes but also that of variable genes led to an increased sensitivity to DNA damage induced by electron beam irradiation. Multiple mutants of ku, ligase or polymerase encoding genes showed an aggravated phenotype compared to single mutants. Biochemical assays revealed the ability of Ku-like proteins to protect and to stimulate ligation of DNA ends. RT-qPCR and GFP fusion experiments suggested that ku-like genes show a growth phase dependent expression profile consistent with their involvement in DNA repair during spores formation and/or germination.
Conteúdo de fenólicos, flavonoides totais e atividade antioxidante de amostras de própolis comerciais

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Elizângela Alves

2013-01-01

Full Text Available Neste trabalho foram determinadas algumas características físico-químicas de própolis de Santa Maria- RS. Foram analisadas seis amostras comerciais obtidas em farmácias e lojas de produtos naturais na cidade citada. A composição química, quanto aos fenólicos e flavonoides totais, assim como a atividade antioxidante, foi testada e comparada. A alta atividade antioxidante dos extratos puros (ou diluídos foi explicada pelo teor dos flavonoides e fenólicos totais, confirmando a relação desses compostos na ação preventiva contra radicais livres. As seis amostras comerciais analisadas possuíram de fenólicos 70,60 mg a 539,10 mg (expresso em mg de ácido gálico/g de extrato de própolis e de flavonoides 48,95 mg a 114,50 mg (expresso em mg de quercetina/g de extrato de própolis. A atividade antioxidante maior foi do extrato aquoso, tanto pela inibição do radical DPPH quanto pela atividade quelante. No extrato aquoso de própolis obteve-se resultados satisfatórios para uma futura aplicação em alimento.
El Frente Estudiantil Nacional (FEN: juventud y estudiantado en el proceso contestatario de los años sesenta en Argentina

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Alejandra Reta

2009-12-01

Full Text Available Este artículo forma parte de un proyecto más amplio que aborda el proceso de acercamiento al Peronismo por parte de un sector del movimiento estudiantil en Argentina durante los años sesentas, tomando el caso particular del Frente Estudiantil Nacional (FEN. El FEN fue una organización universitaria “de pasaje al peronismo”, que buscaba inserción “legítima” en el mismo, vinculándose a sectores del peronismo histórico y al trabajo barrial.Proponemos privilegiar el análisis de documentos, aunque incluimos también algunos testimonios que nos sirven de apoyo. A partir de allí pretendemos explorar algunos “componentes contestatarios”, ligados al clima de ideas de la época, y visualizar cómo este actor colectivo se posiciona dentro de las categorías de “juventud” y del “estudiantado”, que aparecen en el discurso, considerando que el FEN reivindicó constantemente su carácter de tendencia estudiantil universitaria y su militancia juvenil.
Turismo religioso: fenómeno social y económico

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Sandra Maribel Tobón Perilla

2013-12-01

Full Text Available El turismo religioso, como fenómeno social y económico, ha cobrado interés entre académicos y empresarios por las dinámicas que ha generado en las comunidades receptoras de visitantes. Para los primeros, el interés radica en el estudio de las motivaciones, intereses y necesidades espirituales o culturales que parecen haber despertado los centros religiosos; para los segundos, el impacto económico y las oportunidades de rentas adicionales que generan los visitantes, creando nuevas oportunidades de negocios y fuentes de ingresos y empleo para las comunidades aledañas. Este documento hace una revisión a la bibliografía sobre el tema de turismo religioso en el mundo, para lo cual se consultaron las bases de datos ScienceDirect, Jstor, Ebsco, ProQuest y Google Académico, a fin de rastrear las publicaciones más relevantes y estudios empíricos sobre el tema, haciendo un recorrido desde 1963 hasta 2013. Se concluye que el turismo religioso es un fenómeno que tiene su antecedente en el peregrinaje a centros religiosos, pero donde además de las motivaciones, netamente religiosas –ofrecer un sacrificio, obtener un beneficio divino o expiar pecados–, existe un interés cultural, en términos de conocer centros históricos o profundizar sobre las raíces de un credo religioso. Adicionalmente, se observa cómo el turismo religioso se ha convertido en un motor de desarrollo económico de las ciudades receptoras de viajeros a sitios religioso.
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.)
Stream characteristics and their implications for the protection of riparian fens and meadows

DEFF Research Database (Denmark)

Baattrup-Pedersen, A.; Larsen, S.E.; Andersen, Peter Mejlhede

2011-01-01

the influence of stream size, morphology and chemical water characteristics for the distribution of water-dependent terrestrial habitat types, i.e. alkaline fens, periodically inundated meadows and meadows in riparian areas in Denmark using an extensive data set covering a total of 254 stream reaches. A species......1. Running waters, including associated riparian areas, are embraced by international legal frameworks outlining targets for the preservation, protection and improvement of the quality of the environment. Interactions between stream and river processes and riparian habitats have not received much...... attention in the management of stream ecosystems, and integrated measures that consider both the ecological status of streams and rivers (sensu EU Water Framework Directive, WFD) and the conservation status of riparian habitats and species (sensu EU Habitats Directive, HD) are rare. 2. Here, we analysed...
Pressões (sócio)linguísticas na determinação do acento enfático: análise de alguns fenômenos variáveis

OpenAIRE

Carlos Alexandre Gonçalves

2017-01-01

Analise do fenômeno da ênfase prosódica, tomando como base seu efeito em fenômenos de variação/mudança linguística. Descrição da ênfase como grupo de fator relevante em pesquisas labovianas sobre processo de assimilação -ndo/-no. os fenômenos de aférese e prótese em processos de intensificação da língua.
DNA2—An Important Player in DNA Damage Response or Just Another DNA Maintenance Protein?

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Elzbieta Pawłowska

2017-07-01

Full Text Available The human DNA2 (DNA replication helicase/nuclease 2 protein is expressed in both the nucleus and mitochondria, where it displays ATPase-dependent nuclease and helicase activities. DNA2 plays an important role in the removing of long flaps in DNA replication and long-patch base excision repair (LP-BER, interacting with the replication protein A (RPA and the flap endonuclease 1 (FEN1. DNA2 can promote the restart of arrested replication fork along with Werner syndrome ATP-dependent helicase (WRN and Bloom syndrome protein (BLM. In mitochondria, DNA2 can facilitate primer removal during strand-displacement replication. DNA2 is involved in DNA double strand (DSB repair, in which it is complexed with BLM, RPA and MRN for DNA strand resection required for homologous recombination repair. DNA2 can be a major protein involved in the repair of complex DNA damage containing a DSB and a 5′ adduct resulting from a chemical group bound to DNA 5′ ends, created by ionizing radiation and several anticancer drugs, including etoposide, mitoxantrone and some anthracyclines. The role of DNA2 in telomere end maintenance and cell cycle regulation suggests its more general role in keeping genomic stability, which is impaired in cancer. Therefore DNA2 can be an attractive target in cancer therapy. This is supported by enhanced expression of DNA2 in many cancer cell lines with oncogene activation and premalignant cells. Therefore, DNA2 can be considered as a potential marker, useful in cancer therapy. DNA2, along with PARP1 inhibition, may be considered as a potential target for inducing synthetic lethality, a concept of killing tumor cells by targeting two essential genes.
Prospects of fen restoration in relation to changing land use—An example from central Poland

NARCIS (Netherlands)

Klimkowska, A.; Dzierza, P.; Grootjans, A.P.; Kotowski, W.; Diggelen, R.

2010-01-01

We carried out an eco-hydrological analysis to evaluate the most important effects of land use changes on the hydrological functioning of a fen system in Poland. We measured water levels (hydraulic heads) and water flow along a transect through the study area and also analysed land use changes using
Prospects of fen restoration in relation to changing land use-An example from central Poland

NARCIS (Netherlands)

Klimkowska, Agata; Dzierża, Paulina; Grootjans, Ab P.; Kotowski, Wiktor; Diggelen, Rudy Van

2010-01-01

We carried out an eco-hydrological analysis to evaluate the most important effects of land use changes on the hydrological functioning of a fen system in Poland. We measured water levels (hydraulic heads) and water flow along a transect through the study area and also analysed land use changes using
Atividade antioxidante e teor de fenólicos em couve-manteiga (brassica oleracea l. var. acephala submetida a diferentes sistemas de cultivo e métodos de preparo

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Geysa Duarte Junger Rigueira

2017-01-01

Full Text Available O estudo avaliou a influência de sistemas de cultivo e de métodos de preparo na atividade antioxidante e no teor de fenólicos de folhas e talos da couve manteiga (Brassica Oleracea L. var. acephala. Amostras de couve cultivadas pelo sistema convencional e orgânico foram pesadas, higienizadas e os talos separados das folhas. Foram submetidas aos modos de preparo: cru (couve in natura; calor seco (refogada e calor úmido (imersa em água fervente. Avaliou-se à atividade antioxidante e os compostos fenólicos pelo método espectrofotométrico. A verificação de compostos fenólicos e compostos com atividade antioxidante foi realizada por Cromatografia em Camada Delgada (CCD. O sistema de cultivo orgânico e o preparo em calor seco foram os tratamentos que obtiveram os maiores percentuais de antioxidantes e teores de compostos fenólicos, principalmente, em folhas de couve. A atividade antioxidante variou de 38 a 87% nas folhas e de 13 a 56% nos talos de couve. Quanto aos compostos fenólicos os teores médios estiveram entre 173 e 244 mg EAG/100g nas folhas e 86 e 180 mg EAG/100g nos talos. As análises por CCD revelaram que todos os extratos de folhas e talos de couve apresentaram compostos fenólicos bem como componentes com ação antioxidante, mas nem todos com correlação. Conclui-se que a combinação de sistema de cultivo orgânico e cocção em calor seco pode ser eficiente para preservação e ou aumento do teor de compostos fenólicos e da atividade antioxidante em talos e, principalmente, em folhas de couve.
Impacts of road construction and removal on the hydrologic and geochemical function of a fen peatland

Science.gov (United States)

Wells, C. M.; Petrone, R. M.; Sutherland, G.; Price, J. S.

2015-12-01

Linear disturbances such as roads cover vast swaths of northeastern Alberta, the majority of which are wetlands with shallow and local hydrologic connections. Thus, the effects of road construction on wetland hydrological pathways can have significant implications on water movement within the region, and by extension the productivity of vegetation communities and carbon sequestration. However, little is known about the effect that roads have on wetland hydrology. In 2013, a gravel road built within a fen peatland was reclaimed to evaluate hydrologic impacts post removal. Prior to removal, ground and surface water flow was obstructed leading to surface ponding, and vegetation mortality was observed on the up-gradient (wet) side of the road. Rebounding of the peat column was observed throughout the fen immediately following road removal in 2013 (maximum of 12 cm, mean of 2 cm), with modest but slightly smaller expansion in 2014. For both years, peat rebound was greatest in areas where the road was removed. Peat physical properties contrasted sharply between the reclaimed road (RR) peat and the adjacent, unimpacted peatland (UP). Surface bulk densities (pb, 0-10 cm) ranged from 0.1-0.25 g cm-3 along the RR compared to 0.02-0.07 g cm-3 for the UP and on average, pb for all depths were lower at the RR compared to the UP. Similar spatial patterns were observed for peat porosity. Correspondingly low horizontal saturated hydraulic conductivities (Kh) were observed along the RR compared to the UP, averaging 5.7x10-4 m s-1 and 1.7x10-3 m s-1, respectively. The local flow system across the RR and thus subsurface flow was impeded by almost half (0.4 m d-1) compared to flow observed within the UP (0.8 m d-1), leading to ponding on the upgradient side. A marked change in hydrophysical properties and ground and surface water flow patterns post road removal has implications for plant reestablishment and restoration and will form the basis of further study.
The Wor1-like protein Fgp1 regulates pathogenicity, toxin synthesis and reproduction in the phytopathogenic fungus Fusarium graminearum.

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Wilfried Jonkers

Full Text Available WOR1 is a gene for a conserved fungal regulatory protein controlling the dimorphic switch and pathogenicity determents in Candida albicans and its ortholog in the plant pathogen Fusarium oxysporum, called SGE1, is required for pathogenicity and expression of key plant effector proteins. F. graminearum, an important pathogen of cereals, is not known to employ switching and no effector proteins from F. graminearum have been found to date that are required for infection. In this study, the potential role of the WOR1-like gene in pathogenesis was tested in this toxigenic fungus. Deletion of the WOR1 ortholog (called FGP1 in F. graminearum results in greatly reduced pathogenicity and loss of trichothecene toxin accumulation in infected wheat plants and in vitro. The loss of toxin accumulation alone may be sufficient to explain the loss of pathogenicity to wheat. Under toxin-inducing conditions, expression of genes for trichothecene biosynthesis and many other genes are not detected or detected at lower levels in Δfgp1 strains. FGP1 is also involved in the developmental processes of conidium formation and sexual reproduction and modulates a morphological change that accompanies mycotoxin production in vitro. The Wor1-like proteins in Fusarium species have highly conserved N-terminal regions and remarkably divergent C-termini. Interchanging the N- and C- terminal portions of proteins from F. oxysporum and F. graminearum resulted in partial to complete loss of function. Wor1-like proteins are conserved but have evolved to regulate pathogenicity in a range of fungi, likely by adaptations to the C-terminal portion of the protein.
P1 and N170 components distinguish human-like and animal-like makeup stimuli.

Science.gov (United States)

Luo, Shuwei; Luo, Wenbo; He, Weiqi; Chen, Xu; Luo, Yuejia

2013-06-19

This study used event-related potentials to investigate the sensitivity of P1 and N170 components to human-like and animal-like makeup stimuli, which were derived from pictures of Peking opera characters. As predicted, human-like makeup stimuli elicited larger P1 and N170 amplitudes than did animal-like makeup stimuli. Interestingly, a right hemisphere advantage was observed for human-like but not for animal-like makeup stimuli. Dipole source analyses of 130-200-ms window showed that the bilateral fusiform face area may contribute to the differential sensitivity of the N170 component in response to human-like and animal-like makeup stimuli. The present study suggests that the amplitudes of both the P1 and the N170 are sensitive for the mouth component of face-like stimuli.
Análisis de los fenómenos meteorológicos que provocaron penetraciones del mar en el Malecón habanero.

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Lázara Díaz Maqueira

2003-03-01

Full Text Available Se presenta la caracterización de algunos de los fenómenos meteorológicos peligrosos que produjeron penetraciones del mar en el Malecón habanero y su tipificación sinóptica, con el objetivo de que se utilice como herramienta de trabajo para predecir con suficiente antelación este fenómeno y de esta forma disminuir las consecuencias del mismo en el área urbana aledaña. También se realiza una descripción del Malecón habanero por tramos y los factores que producen las inundaciones costeras por penetraciones del mar. La comprensión de las interacciones entre las características constructivas del Malecón y los factores desencadenantes de las inundaciones han ayudado a predecir la magnitud del fenómeno y alertar convenientemente a las organizaciones estatales e intereses costeros para que tomen las medidas requeridas en cada caso.
Mastermind-Like 1 Is Ubiquitinated: Functional Consequences for Notch Signaling.

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Mozhgan Farshbaf

Full Text Available Early studies demonstrated the involvement of ubiquitination of the Notch intracellular domain for rapid turnover of the transcriptional complex at Notch target genes. It was shown that this ubiquitination was promoted by the co-activator Mastermind like 1 (MAML1. MAML1 also contains numerous lysine residues that may also be ubiquitinated and necessary for protein regulation. In this study, we show that over-expressed MAML1 is ubiquitinated and identify eight conserved lysine residues which are required for ubiquitination. We also show that p300 stimulates ubiquitination and that Notch inhibits ubiquitination. Furthermore, we show that a mutant MAML1 that has decreased ubiquitination shows increased output from a HES1 reporter gene assay. Therefore, we speculate that ubiquitination of MAML1 might be a mechanism to maintain low levels of the protein until needed for transcriptional activation. In summary, this study identifies that MAML1 is ubiquitinated in the absence of Notch signaling to maintain low levels of MAML1 in the cell. Our data supports the notion that a precise and tight regulation of the Notch pathway is required for this signaling pathway.
Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase.

Science.gov (United States)

Kopera, Huira C; Moldovan, John B; Morrish, Tammy A; Garcia-Perez, Jose Luis; Moran, John V

2011-12-20

Long interspersed element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that contain activities required for conventional retrotransposition by a mechanism termed target-site primed reverse transcription. Previous experiments in XRCC4 or DNA protein kinase catalytic subunit-deficient CHO cell lines, which are defective for the nonhomologous end-joining DNA repair pathway, revealed an alternative endonuclease-independent (ENi) pathway for L1 retrotransposition. Interestingly, some ENi retrotransposition events in DNA protein kinase catalytic subunit-deficient cells are targeted to dysfunctional telomeres. Here we used an in vitro assay to detect L1 reverse transcriptase activity to demonstrate that wild-type or endonuclease-defective L1 ribonucleoprotein particles can use oligonucleotide adapters that mimic telomeric ends as primers to initiate the reverse transcription of L1 mRNA. Importantly, these ribonucleoprotein particles also contain a nuclease activity that can process the oligonucleotide adapters before the initiation of reverse transcription. Finally, we demonstrate that ORF1p is not strictly required for ENi retrotransposition at dysfunctional telomeres. Thus, these data further highlight similarities between the mechanism of ENi L1 retrotransposition and telomerase.

Relación entre el fenómeno de la ilusión de la mano falsa y variables personales

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Gina Rodríguez-Melgarejo

2015-07-01

Full Text Available El fenómeno de la ilusión de la mano falsa (IMF se ha propuesto como un diseño que permite discriminar procesos perceptuales a partir de entradas sensoriales multimodales. Se han reportado varias modificaciones y variables que podrían afectar la generación del fenómeno. Este estudio se planteó bajo un diseño descriptivo-correlacional del fenómeno. Se realizó el procedimiento en jóvenes (16-48 años M = 20.86 años, DE = 4.4 colombianos para replicar el fenómeno de IMF. Los sujetos se dividieron al azar en dos grupos que diferían en el tipo de estimulación (sincrónica y desincronizada. La identificación de la IMF se realizó mediante la escala reportada en múltiples estudios, mostrando que en algunas variables como la estimulación se presentaba una diferencia en el reporte de IMF (P2. χ2 = 5.856: p < 0.05; P3. χ2 = 6.655: p < 0.01 y P7 (χ2 = 4.764: p < 0.05. Otras variables como sexo y dominancia manual mostraron algunas diferencias. La escala presenta una consistencia interna aceptable (α = 0.8. Los resultados de la IMF son similares a algunos estudios pero con puntuaciones menores que en los estudios iniciales, la diferencia en cuanto al género en tareas perceptuales podría explicar gran parte de los resultados.
BOREAS TGB-3 Plant Species Composition Data over the NSA Fen

Science.gov (United States)

Bubier, Jill L.; Hall, Forrest G. (Editor); Conrad, Sara K. (Editor)

2000-01-01

The BOReal Ecosystem-Atmosphere Study Trace Gas Biogeochemistry (BOREAS TGB-3) team collected several data sets that contributed to understanding the measured trace gas fluxes over sites in the Northern Study Area (NSA). This data set contains information about the composition of plant species that were within the collars used to measure Net Ecosystem Exchange of CO2 (NEE). The species composition was identified to understand the differences in NEE among the various plant communities in the NSA fen. The data were collected in July of 1994 and 1996. The data are contained in comma-delimited, ASCII files.
Cyberbullying: percepções acerca do fenômeno e das estratégias de enfrentamento

Directory of Open Access Journals (Sweden)

Sidclay Bezerra Souza

2014-01-01

Full Text Available O cyberbullying, caracterizado como um dos tipos de violência em contexto educativo, apresenta-se como uma das principais causas do mal-estar vivido por seus protagonistas. O estudo inserido no Projeto Cyberbullying - o diagnóstico da situação em Portugal pretendeu compreender o fenômeno a partir das vivências de 118 estudantes do primeiro ano do Ensino Superior público português, que responderam a um questionário. Pudemos perceber a percepção dos alunos sobre o fenômeno e a importância que pares, professores e pais apresentam no enfrentamento e prevenção do cyberbullying. Tais resultados orientam-se no sentido do desenvolvimento sistêmico de comunidades que se fortalecem internamente para que cada um se sinta autorizado a intervir e a cuidar dos que nelas são vítimas de violência.
Turn on, fade out - methane exchange in a coastal fen over a period of six years after rewetting

Science.gov (United States)

Jurasinski, Gerald; Glatzel, Stephan; Hahn, Juliane; Koch, Stefan; Koch, Marian; Koebsch, Franziska

2016-04-01

The rewetting of drained peatlands is widely regarded as an adequate measure for the mitigation of greenhouse gas emissions. Therefore, especially in NE Germany, many peatlands are being rewetted. Our knowledge about greenhouse gas exchange associated with rewetting is mainly based on short-term experiments or space-for-time substitutions. These approaches do not consider the transient character of ecosystem acclimatization to flooding by rewetting. Moreover data in this regard on coastal peatland ecosystems are sparse. Here, we present 7 years of data on CH4-exchange in a coastal fen after rewetting by flooding. On the site „Rodewiese", which is located within the NSG "Hütelmoor und Heiligensee" in the Northeast of Rostock, NE Germany, we have established a long term research observatory addressing atmospheric C-exchange. The site is part of the TERENO network. Since summer 2009 we determine CH4 fluxes with closed chambers distributed widely across the study site and CO2-exchange with eddy covariance as well as ancillary data on vegetation, hydrology, and biogeochemistry. This talk addresses the CH4-exchange over time whereas CO2-exchange data are presented by Koebsch et al. in the same session. Rewetting turned the site from a summer dry fen with mean annual water levels of around -0.08m into a shallow lake with water levels up to 0.60m. In the first year after flooding, we observed a substantial die-back of vegetation, especially in stands of Carex acutiformis. Flooding increased methane release rates to extremely high levels of up to 4.3 t ha-1 a-1 for sedge stands and 2.7 t ha-1 a-1 on average, which amounts to 75.6 t ha-1 a-1 in CO2-equivalents. Thereafter, the averaged annual CH4 emissions decreased asymptotically and where at an average of 0.5 t ha-1 a-1 (14 t ha-1 a-1 in CO2-equivalents) in 2015. Factoring in the NEE of the growing season (from Eddy measurements) suggests that the system may be slightly above neutral with respect to the greenhouse
Hydrometeorological conditions preceding wildfire, and the subsequent burning of a fen watershed in Fort McMurray, Alberta, Canada

Science.gov (United States)

Elmes, Matthew C.; Thompson, Dan K.; Sherwood, James H.; Price, Jonathan S.

2018-01-01

The destructive nature of the ˜ 590 000 ha Horse river wildfire in the Western Boreal Plain (WBP), northern Alberta, in May of 2016 motivated the investigation of the hydrometeorological conditions that preceded the fire. Historical climate and field hydrometeorological data from a moderate-rich fen watershed were used to (a) identify whether the spring 2016 conditions were outside the range of natural variability for WBP climate cycles, (b) explain the observed patterns in burn severity across the watershed, and (c) identify whether fall and winter moisture signals observed in peatlands and lowland forests in the region are indicative of wildfire. Field hydrometeorological data from the fen watershed confirmed the presence of cumulative moisture deficits prior to the fire. Hydrogeological investigations highlighted the susceptibility of fen and upland areas to water table and soil moisture decline over rain-free periods (including winter), due to the watershed's reliance on supply from localized flow systems originating in topographic highs. Subtle changes in topographic position led to large changes in groundwater connectivity, leading to greater organic soil consumption by fire in wetland margins and at high elevations. The 2016 spring moisture conditions measured prior to the ignition of the fen watershed were not illustrated well by the Drought Code (DC) when standard overwintering procedures were applied. However, close agreement was found when default assumptions were replaced with measured duff soil moisture recharge and incorporated into the overwintering DC procedure. We conclude that accumulated moisture deficits dating back to the summer of 2015 led to the dry conditions that preceded the fire. The infrequent coinciding of several hydrometeorological conditions, including low autumn soil moisture, a modest snowpack, lack of spring precipitation, and high spring air temperatures and winds, ultimately led to the Horse river wildfire spreading widely and
Saturated and unsaturated salt transport in peat from a constructed fen

Science.gov (United States)

Simhayov, Reuven B.; Weber, Tobias K. D.; Price, Jonathan S.

2018-02-01

transport using Hydrus-1-D. The simulation showed a good prediction of observed values, confirming the suitability of the parameters for use in a slightly unsaturated transport simulation. The findings improve the understanding of solute redistribution in the constructed fen and imply that MIM should not be automatically assumed for solute transport in peat but rather should be evidence based.
Phase Transformations and Phase Equilibria in the Fe-N System at Temperatures below 573 K

DEFF Research Database (Denmark)

Malinov, S.; Böttger, A.J.; Mittemeijer, E.J.

2001-01-01

The phase transformations of homogeneous Fe-N alloys of nitrogen contents from 10 to 26 at. pct were investigated by means of X-ray diffraction analysis upon aging in the temperature range from 373 to 473 K. It was found that precipitation of alpha double prime-Fe16N2 below 443 K does not only oc...
Regulation of abiotic stress signalling by Arabidopsis C-terminal domain phosphatase-like 1 requires interaction with a k-homology domain-containing protein.

Directory of Open Access Journals (Sweden)

In Sil Jeong

Full Text Available Arabidopsis thaliana CARBOXYL-TERMINAL DOMAIN (CTD PHOSPHATASE-LIKE 1 (CPL1 regulates plant transcriptional responses to diverse stress signals. Unlike typical CTD phosphatases, CPL1 contains two double-stranded (ds RNA binding motifs (dsRBMs at its C-terminus. Some dsRBMs can bind to dsRNA and/or other proteins, but the function of the CPL1 dsRBMs has remained obscure. Here, we report identification of REGULATOR OF CBF GENE EXPRESSION 3 (RCF3 as a CPL1-interacting protein. RCF3 co-purified with tandem-affinity-tagged CPL1 from cultured Arabidopsis cells and contains multiple K-homology (KH domains, which were predicted to be important for binding to single-stranded DNA/RNA. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation analyses established that CPL1 and RCF3 strongly associate in vivo, an interaction mediated by the dsRBM1 of CPL1 and the KH3/KH4 domains of RCF3. Mapping of functional regions of CPL1 indicated that CPL1 in vivo function requires the dsRBM1, catalytic activity, and nuclear targeting of CPL1. Gene expression profiles of rcf3 and cpl1 mutants were similar during iron deficiency, but were distinct during the cold response. These results suggest that tethering CPL1 to RCF3 via dsRBM1 is part of the mechanism that confers specificity to CPL1-mediated transcriptional regulation.
Tousled-like kinases phosphorylate Asf1 to promote histone supply during DNA replication

DEFF Research Database (Denmark)

Klimovskaia, Ilnaz M; Young, Clifford; Strømme, Caroline B

2014-01-01

During DNA replication, nucleosomes are rapidly assembled on newly synthesized DNA to restore chromatin organization. Asf1, a key histone H3-H4 chaperone required for this process, is phosphorylated by Tousled-like kinases (TLKs). Here, we identify TLK phosphorylation sites by mass spectrometry...
Cell-based delivery of glucagon-like peptide-1 using encapsulated mesenchymal stem cells

DEFF Research Database (Denmark)

Wallrapp, Christine; Thoenes, Eric; Thürmer, Frank

2013-01-01

Glucagon-like peptide-1 (GLP-1) CellBeads are cell-based implants for the sustained local delivery of bioactive factors. They consist of GLP-1 secreting mesenchymal stem cells encapsulated in a spherically shaped immuno-isolating alginate matrix. A highly standardized and reproducible encapsulation...... and quality control is performed in compliance with good manufacturing practice and fulfils all regulatory requirements for human clinical use. GLP-1 CellBeads combine the neuro- and cardioprotective properties of both GLP-1 and mesenchymal stem cells. First promising results were obtained from preclinical...... method is described for the manufacturing of homogeneous CellBeads. Viability and sustained secretion was shown for the recombinant GLP-1 and the cell endogenous bioactive factors like vascular endothelial growth factor, neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor. Manufacturing...
Areva: 1. quarter 2015 revenue down, at euros 1.762 bn: -1.1% vs. March 2014 (-0.9% like for like)

International Nuclear Information System (INIS)

Repaire, Philippine du

2015-01-01

In the 1. quarter of 2015, AREVA generated consolidated revenue of 1.762 billion euros, representing a decrease of 1.1% (-0.9% like for like) compared with the same period in 2014. Foreign exchange had a positive impact of 36 million euros over the period, while consolidation scope had a negative impact of 39 million euros. At March 31, 2015, the group had 47.520 billion euros in backlog, a 1.4% increase in relation to December 31, 2014 (46.866 billion euros) reflecting a favorable foreign exchange impact. It should be noted that the backlog does not include the amount from agreements signed with EDF in October 2013 for the EPR reactors project at Hinkley Point in the United Kingdom or for the related fuel. The order intake totaled 881 million euros in the 1. quarter of 2015, an increase compared with the 1. quarter of 2014 (668 million euros)
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)
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.
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.
La definición jurídica del fenómeno metropolitano en el ámbito subnacional mexicano

Directory of Open Access Journals (Sweden)

Alberto Arellano Ríos

2014-10-01

Full Text Available En este texto se analiza la definición jurídica del fenómeno metropolitano en seis Estados de la república mexicana. Se estudian seis legislaciones estatales creadas expresamente para atender y darle forma jurídica a un problema que se ha acentuado con mayor fuerza en México, el cual desde el punto de vista de las instancias gubernamentales tiene mayor relevancia en los últimos veinte años. De este modo, el documento esboza el fenómeno metropolitano como contexto social. Luego traza los mecanismos constitucionales contemplados para gestionar y gobernar las zonas metropolitanos para, finalmente, exponer los casos estatales que ante la ausencia de un marco legal nacional, y desde la lógica del sistema federal, intentan sentar bases jurídicas para resolver este problema.
Multiple requirements of PLK1 during mouse oocyte maturation.

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Petr Solc

Full Text Available Polo-like kinase 1 (PLK1 orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1's functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC. Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.
Effectiveness of turf stripping as a measure for restoring species-rich fen meadows in suboptimal hydrological conditions

NARCIS (Netherlands)

Hoek, van der D.; Heijmans, M.M.P.D.

2007-01-01

Most species-rich fen meadows in nature reserves in The Netherlands are acidified due to weaker upwelling of base-rich groundwater. The present study investigated whether and why turf stripping combined with superficial drainage might promote the long-term recovery of such meadows and restore the
Formation cross section of iron-60 with reactor neutrons in 59Fe(n, γ)60Fe reaction

International Nuclear Information System (INIS)

Sato, T.; Suzuki, T.

1993-01-01

Ingrowth of 60 Co radioactivity in an iron sample irradiated in a nuclear reactor has been measured for determination of formation cross section of 60 Fe in the 59 Fe(n, γ) 60 Fe reaction with reactor neutrons. After 5 years cooling, the irradiated iron was purified from 60 Co and other radioactive nuclides by an anion exchange separation method and isopropyl ether extraction in hydrochloric acid. The amount of 60 Co ingrowth was measured by γ-spectrometry using a Ge-detector coupled to a multichannel pulse height analyzer 4 years after the purification of iron. Neutron flux of the irradiation position was calculated from the amount of 55 Fe produced. The observed value of 12.5 ± 2.8 barn is slightly greater than reported value for burnup cross section of 59 Fe(n, x)X, where x refers γ, α, d, p and 2n, and X is any nuclide produced by the above reactions. (author) 8 refs.; 2 tabs
Jogos de Empresas: Abordagens ao Fenômeno, Perspectivas Teóricas e Metodológicas

Directory of Open Access Journals (Sweden)

Valdete de Oliveira Mrtvi

2017-01-01

Full Text Available Os jogos de empresas, além de utilizados no processo de ensino, também são explorados como ferramentas de pesquisa, conforme Gentry, Tice, Robertson e Gentry (1984. Este estudo, de natureza qualitativa e quantitativa, descritivo quanto aos objetivos, e caracterizado como revisão integrativa no tocante ao método, trata do seguinte problema de pesquisa: Qual o perfil da pesquisa acadêmica brasileira sobre jogos de empresas considerando a abordagem ao fenômeno e as perspectivas teóricas e metodológicas? Foram analisados 226 artigos extraídos de periódicos online, do indexador Spell e anais de eventos. Como resultado, é proposta uma tipologia para classificação das pesquisas em jogos de empresas, considerando a abordagem ao fenômeno de pesquisa e os eixos temáticos. Constatou-se a predominância de estudos que têm as simulações como objeto de pesquisa - SCOPE (77,9% -, enquanto 22,1% utilizam os jogos de empresas como ambiente de pesquisa - SCAPE. Verificou-se que 38,5% dos estudos não mencionam quaisquer das classificações metodológicas analisadas. Discute-se algumas fragilidades metodológicas identificadas nas pesquisas da área. Apesar de as classificações não serem determinantes para a qualidade de um estudo, representam importante indicador da consistência do processo de reflexão e da coesão da investigação.

Fenótipo cintura hipertrigliceridêmica: associação com alterações metabólicas em adolescentes Hypertriglyceridemic waist phenotype: association with metabolic abnormalities in adolescents

Directory of Open Access Journals (Sweden)

Maria Ester P. da Conceição-Machado

2013-02-01

Full Text Available OBJETIVO: O presente estudo objetivou identificar a prevalência do fenótipo cintura hipertrigliceridêmica (CHT e avaliar sua associação com alterações metabólicas em adolescentes de baixa condição econômica. MÉTODO: Estudo transversal com amostra probabilística de 1.076 adolescentes entre 11 e 17 anos, de ambos os sexos, estudantes de escolas públicas. Os participantes foram submetidos à avaliação antropométrica (peso, altura e circunferência da cintura e à dosagem dos níveis de colesterol total, LDL-C, HDL-C, colesterol não HDL, triglicérides (TG e glicemia de jejum. Foram obtidas informações referentes às condições econômicas das famílias dos participantes.O fenótipo CHT foi definido pela presença simultânea da circunferência da cintura aumentada (> percentil 90 por idade e sexo e dos níveis séricos de triglicérides elevados (> 100 mg/dL. A análise de regressão logística foi utilizada para avaliação das associações de interesse. RESULTADOS: A prevalência do fenótipo CHT foi de 7,2% entre os adolescentes, sendo mais elevada na presença de obesidade (63,4%, do colesterol não HDL (16,6% e do LDL-C (13,7% altos. A análise bivariada indicou que, das variáveis metabólicas, apenas a glicemia não se associou ao fenótipo CHT. A análise multivariada, ajustada por sexo e idade, indicou que o fenótipo CHT se associou positivamente com o colesterol não HDL alto (odds ratio, 7,0; IC 95% 3,9-12,6 e com o HDL-C baixo (odds ratio, 2,7; IC 95%, 1,5-4,8. CONCLUSÕES: Este estudo mostrou que o fenótipo CHT se associou com um perfil lipídico aterogênico e sugere esse fenótipo como uma ferramenta de screening que pode ser utilizada para identificar adolescentes com alterações metabólicas.OBJECTIVE: This study aimed to identify the prevalence of hypertriglyceridemic waist (HTW phenotype, and to evaluate its association with metabolic abnormalities in adolescents of low socioeconomic status. METHOD: This
De columbine à virgínia tech: reflexões com base empírica sobre um fenômeno em expansão

OpenAIRE

Vieira,Timoteo Madaleno; Mendes,Francisco Dyonísio Cardoso; Guimarães,Leonardo Conceição

2009-01-01

O fenômeno conhecido como school shooting vem chamando a atenção do mundo, principalmente após a tragédia na Columbine High School, na cidade de Littleton, Colorado, nos Estados Unidos, em 1999. Posteriormente, em abril de 2007, o estudante Cho Seung-Hui protagonizou um massacre de proporções ainda maiores ao atirar contra estudantes do Virginia Polytechnic Institute and State University, matando 32 pessoas e se suicidando ao final. O presente artigo reflete sobre o fenômeno chamando a atençã...
FEN LABORATUARLARININ İŞLEVSELLİĞİNİN İLKÖĞRETİM OKULLARINDA DEĞERLENDİRİLMESİ

OpenAIRE

YILAYAZ, Ömer; TURAN, Mehmet; BAHŞİ, Muammer

2009-01-01

Bu araştırmada, ilköğretim okullarındaki fen laboratuarlarının işlevselliğine ilişkin öğrenci görüşlerinin belirlemesi amaçlanmıştır. Bu amaçla 19 maddelik bir anket formu hazırlanmıştır. Çalışma, Fırat Üniversitesi Eğitim Fakültesi Fen ve Teknoloji öğretmenliği bölümünde öğrenim gören 76 öğrenciye (4. sınıf) uygulanmıştır. Anket sonucunda elde edilen veriler, SPSS paket istatistik programında değerlendirilmiştir.
Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability

KAUST Repository

Tsutakawa, Susan E.

2017-06-27

DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5\\'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5\\'-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5\\'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via phosphate steering\\', basic residues energetically steer an inverted ss 5\\'-flap through a gateway over FEN1\\'s active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA) repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5\\'-flap specificity and catalysis, preventing genomic instability.
Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells

Directory of Open Access Journals (Sweden)

Chad M. Toledo

2015-12-01

Full Text Available To identify therapeutic targets for glioblastoma (GBM, we performed genome-wide CRISPR-Cas9 knockout (KO screens in patient-derived GBM stem-like cells (GSCs and human neural stem/progenitors (NSCs, non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers. In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality.
Effect of the processing in the phenolic compounds content and antioxidant activity in apple wine/ Efeito do processamento no teor de compostos fenólicos e na atividade antioxidante em fermentados de maçã

Directory of Open Access Journals (Sweden)

Alessandro Nogueira

2008-08-01

Full Text Available In the apple processing the phenolic compounds play a remarkable role in sensorial and functional aspects like color and flavor, and antioxidant activity, respectively. This work was done aiming to evaluate the effect of the different operations and the alcoholic fermentation in the phenol content and the antioxidant activity in apple wine processing. The phenol content of apple must without enzymatic oxidation was 1085±265; 958±94 and 787±64 mg.L-1 for the varieties Gala, Joaquina and Fuji, respectively. After the despectinization and filtration step, with total oxidation, the values had been reduced for 451±6.4; 408±4.2 and 255±9.5 mg.L-1. The losses were observed up to 9 hours of fermentation and after this period the values of phenols remain steady until the end of the process (360 hours. The antioxidant activity showed similar effect, with an initial content in apple must without enzymatic oxidation of 10807±1530; 8473±853 and 6449±1272 µM for Gala, Fuji and Joaquina, and after the despectinização and filtration with enzymatic oxidation, the values decreased to 4376±63; 1909±84 and 2889±55 µM. The main losses of phenolics compounds and antioxidant activity in the fermentation processing of apple must occurred during the reaction of enzymatic browning within the operations of depectinization and filtration.No processamento de maçã, os compostos fenólicos desempenham papel fundamental em aspectos sensoriais, como cor e sabor, e em aspectos funcionais em virtude da atividade antioxidante. O trabalho teve como objetivo avaliar o efeito das operações unitárias e da fermentação alcoólica no teor de fenóis e na atividade antioxidante de fermentados de maçã. Os teores de fenóis do mosto após a prensagem, sem oxidação enzimática, foram 1085±265; 958±94 e 787±64 mg.L-1 para as cultivares Gala, Joaquina e Fuji, respectivamente. Após a despectinização e filtração, com oxidação enzimática, os valores diminu
Fenóis totais, atividade antioxidante e constituintes químicos de extratos de Anacardium occidentale L., Anacardiaceae

Directory of Open Access Journals (Sweden)

Mariana H. Chaves

Full Text Available O presente artigo relata o isolamento e identificação do palmitato, oleato e linoleato de sitosterila, sitosterol, estigmasterol, 3-O-β-D-galactopiranosídeo do sitosterol, 3-O-β-Dgalactopiranosídeo do estigmasterol, 3-O-β-D-glicopiranosídeo do sitosterol e uma mistura de ácidos anacárdicos (monoeno e dieno do extrato etanólico de cascas do caule de Anacardium occidentale L., Anacardiaceae, bem como do sitosterol, estigmasterol, lupeol, β-amirina, catequina e epicatequina do extrato etanólico do tegumento da castanha de caju in natura. Os extratos EtOH da casca e do tegumento foram avaliados quanto ao conteúdo de fenóis totais e atividade antioxidante. O extrato etanólico das cascas do caule apresentou maior conteúdo de compostos fenólicos e percentual de atividade antioxidante.
The COMPASS-Like Complex Promotes Flowering and Panicle Branching in Rice1[OPEN

Science.gov (United States)

Wang, Shiliang; Jiang, Haiyang; Cheng, Beijiu

2018-01-01

Flowering time (heading date) and panicle branch number are important agronomic traits that determine yield in rice (Oryza sativa). The activation of flowering requires histone methylation, but the roles of trimethylation of Lys 4 of histone 3 (H3K4me3) in modulating heading date and panicle development are unclear. Here, we showed that the COMPASS-like complex promotes flowering and panicle branching. The rice (Oryza sativa) WD40 protein OsWDR5a interacts with the TRITHORAX-like protein OsTrx1/SET domain group protein 723 (SDG723) to form the core components of the COMPASS-like complex. Plants in which OsWDR5a or OsTrx1 expression was decreased by RNA interference produced fewer secondary branches and less grain and exhibited a delayed heading date under long-day and short-day conditions, whereas loss of OsWDR5a function resulted in embryo lethality. OsWDR5a binds to Early heading date 1 to regulate its H3K4me3 and expression levels. Together, our results show that the COMPASS-like complex promotes flowering and panicle development and suggest that modulation of H3K4me3 levels by the COMPASS-like complex is critical for rice development. PMID:29440594
Identification of flap structure-specific endonuclease 1 as a factor involved in long-term memory formation of aversive learning.

Science.gov (United States)

Saavedra-Rodríguez, Lorena; Vázquez, Adrinel; Ortiz-Zuazaga, Humberto G; Chorna, Nataliya E; González, Fernando A; Andrés, Lissette; Rodríguez, Karen; Ramírez, Fernando; Rodríguez, Alan; Peña de Ortiz, Sandra

2009-05-06

We previously proposed that DNA recombination/repair processes play a role in memory formation. Here, we examined the possible role of the fen-1 gene, encoding a flap structure-specific endonuclease, in memory consolidation of conditioned taste aversion (CTA). Quantitative real-time PCR showed that amygdalar fen-1 mRNA induction was associated to the central processing of the illness experience related to CTA and to CTA itself, but not to the central processing resulting from the presentation of a novel flavor. CTA also increased expression of the Fen-1 protein in the amygdala, but not the insular cortex. In addition, double immunofluorescence analyses showed that amygdalar Fen-1 expression is mostly localized within neurons. Importantly, functional studies demonstrated that amygdalar antisense knockdown of fen-1 expression impaired consolidation, but not short-term memory, of CTA. Overall, these studies define the fen-1 endonuclease as a new DNA recombination/repair factor involved in the formation of long-term memories.
Genome Editing in Human Pluripotent Stem Cells.

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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.
Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3).

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Mulepati, Sabin; Bailey, Scott

2011-09-09

RNA transcribed from clustered regularly interspaced short palindromic repeats (CRISPRs) protects many prokaryotes from invasion by foreign DNA such as viruses, conjugative plasmids, and transposable elements. Cas3 (CRISPR-associated protein 3) is essential for this CRISPR protection and is thought to mediate cleavage of the foreign DNA through its N-terminal histidine-aspartate (HD) domain. We report here the 1.8 Å crystal structure of the HD domain of Cas3 from Thermus thermophilus HB8. Structural and biochemical studies predict that this enzyme binds two metal ions at its active site. We also demonstrate that the single-stranded DNA endonuclease activity of this T. thermophilus domain is activated not by magnesium but by transition metal ions such as manganese and nickel. Structure-guided mutagenesis confirms the importance of the metal-binding residues for the nuclease activity and identifies other active site residues. Overall, these results provide a framework for understanding the role of Cas3 in the CRISPR system.
The climate warming effect of a fen peat meadow with fluctuating water table is reduced by young alder trees

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V. Huth

2018-03-01

Full Text Available Black alder (Alnus glutinosa (L. Gaertn. occurs naturally in temperate marshes and in minerotrophic peatlands and is also suitable for paludiculture - the cultivation of biomass on wet or rewetted peatland. We investigated the effect of a newly established black alder plantation on the greenhouse gas (GHG balance of a degraded fen in north-eastern Germany over a two-year period (August 2010–August 2012. We compared the alder plantation (Awet with an extensively used meadow (Mwet and a drier reference meadow (Mmoist. GHG fluxes were measured monthly to bi-monthly using the closed chamber method. Our results show that Awet was a slight net GHG (in CO2-eq sink of 3.4 ± 1.7 t ha-1 yr-1, Mwet was a moderate net GHG source of 9.6 ± 1.2 t ha 1 yr-1, and Mmoist was a strong net GHG source of 24.5 ± 1.6 t ha-1 yr-1. This was mainly driven by CO2 uptake at the two very moist (wet sites and by high CO2 release at the drier reference site. Awet was a larger CO2 sink than Mwet, probably due to additional CO2 uptake by the alder stand at Awet and carbon export in plant material harvested from Mwet. All sites were significant CH4 sources. Substantial CH4 emission peaks were observed at all sites following extraordinarily heavy precipitation during the summer of 2011, which accounted for up to 70 % of the accumulated two-year CH4 emissions. However, the Awet site generally emitted less CH4, possibly due to the effective oxygen transport mechanism in black alders. N2O emissions were negligible at all three sites. Our results indicate that the GHG balances of formerly drained fens benefit in the short term from planting of black alders, mostly due to reduced CH4 emissions. This study highlights the importance of acknowledging extreme precipitation events and groundwater fluctuations for the derivation of reliable GHG emission factors.
Compostos fenólicos e capacidade antioxidante de cultivares de uvas Vitis labrusca L. e Vitis vinifera L. Phenolic compounds and antioxidant activity of Vitis labrusca and Vitis vinifera cultivars

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Lucile Tiemi Abe

2007-06-01

Full Text Available O conteúdo de compostos fenólicos, incluindo resveratrol, antocianinas e outros flavonóides, e a capacidade antioxidante de cinco cultivares de uvas produzidas em Minas Gerais foram determinados. O conteúdo de fenólicos totais, determinado através do método de Folin-Ciocalteau variou significativamente, entre 65 ± 1 e 390 ± 30 mg equivalentes de ácido gálico.100 g -1 de amostra base úmida (b.u.. O conteúdo de antocianinas totais quantificado por CLAE variou entre 6,7 ± 0,2 e 154 ± 4 mg equivalentes de cianidina.100 g -1 (b.u.. Outros flavonóides encontrados foram catequina, epicatequina, quercetina, caempferol além dos ácidos hidroxicinâmicos. O resveratrol foi encontrado em três cultivares, variando entre 0,022 ± 0,001 e 0,60 ± 0,03 mg.100 g -1 (b.u.. A capacidade antioxidante foi analisada usando o método de seqüestro de radicais livres do DPPH (2,2-difenil-1-picrilhidrazila, que variou entre 2,7 ± 0,1 e 19 ± 2 µmoles equivalentes de Trolox.g -1 de amostra (b.u., mostrando boa correlação com o conteúdo de fenólicos totais. As uvas tintas de coloração mais escura apresentaram maior conteúdo de antocianinas, e conseqüentemente maior teor de fenólicos totais e capacidade antioxidante.Phenolic compounds including resveratrol, anthocyanins and other flavonoids and antioxidant capacity were evaluated for five grape cultivars cultivated in Minas Gerais (Brazil. The content of total phenolic determined by the Folin Ciocalteau method varied significantly, from 65 ± 1 to 390 ± 30 mg gallic acid equivalents.100 g -1 of FW (Fresh Weight. The content of total anthocyanins quantified by HPLC varied from 6.7 ± 0.2 to 154 ± 4 mg of cyanidin equivalents.100 g -1 FW. Resveratrol was found in three cultivars, in contents from 0.022 ± 0.001 to 0.60 ± 0.03 mg.100 g¹ of FW. In addition to hydroxicynnamic acids, other flavonoids such as catechin, epicatechin, quercetin and kaempferol were also present. The antioxidant
Generation of SNCA Cell Models Using Zinc Finger Nuclease (ZFN) Technology for Efficient High-Throughput Drug Screening.

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Dansithong, Warunee; Paul, Sharan; Scoles, Daniel R; Pulst, Stefan M; Huynh, Duong P

2015-01-01

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by loss of dopaminergic neurons of the substantia nigra. The hallmark of PD is the appearance of neuronal protein aggregations known as Lewy bodies and Lewy neurites, of which α-synuclein forms a major component. Familial PD is rare and is associated with missense mutations of the SNCA gene or increases in gene copy number resulting in SNCA overexpression. This suggests that lowering SNCA expression could be therapeutic for PD. Supporting this hypothesis, SNCA reduction was neuroprotective in cell line and rodent PD models. We developed novel cell lines expressing SNCA fused to the reporter genes luciferase (luc) or GFP with the objective to enable high-throughput compound screening (HTS) for small molecules that can lower SNCA expression. Because SNCA expression is likely regulated by far-upstream elements (including the NACP-REP1 located at 8852 bp upstream of the transcription site), we employed zinc finger nuclease (ZFN) genome editing to insert reporter genes in-frame downstream of the SNCA gene in order to retain native SNCA expression control. This ensured full retention of known and unknown up- and downstream genetic elements controlling SNCA expression. Treatment of cells with the histone deacetylase inhibitor valproic acid (VPA) resulted in significantly increased SNCA-luc and SNCA-GFP expression supporting the use of our cell lines for identifying small molecules altering complex modes of expression control. Cells expressing SNCA-luc treated with a luciferase inhibitor or SNCA siRNA resulted in Z'-scores ≥ 0.75, suggesting the suitability of these cell lines for use in HTS. This study presents a novel use of genome editing for the creation of cell lines expressing α-synuclein fusion constructs entirely under native expression control. These cell lines are well suited for HTS for compounds that lower SNCA expression directly or by acting at long-range sites to the SNCA
El fenómeno del bullying: una comparación de dos protocolos de intervención en el País Vasco

OpenAIRE

Goirigolzarri-Sáez, Sandra

2015-01-01

Este trabajo tiene como objetivo abordar el fenómeno del bullying mediante la comparación de dos protocolos de actuación llevados a cabo en el País Vasco para intervenir en esta problemática. Para la consecución de este fin, se ha dividido el marco teórico en dos partes: en la primera, se abordan los aspectos más importantes dentro del fenómeno del bullying como son su definición y características, el perfil de los participantes, las consecuencias psicológicas, el rol de la fam...
FAN1 acts with FANCI-FANCD2 to promote DNA interstrand cross-link repair.

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Liu, Ting; Ghosal, Gargi; Yuan, Jingsong; Chen, Junjie; Huang, Jun

2010-08-06

Fanconi anemia (FA) is caused by mutations in 13 Fanc genes and renders cells hypersensitive to DNA interstrand cross-linking (ICL) agents. A central event in the FA pathway is mono-ubiquitylation of the FANCI-FANCD2 (ID) protein complex. Here, we characterize a previously unrecognized nuclease, Fanconi anemia-associated nuclease 1 (FAN1), that promotes ICL repair in a manner strictly dependent on its ability to accumulate at or near sites of DNA damage and that relies on mono-ubiquitylation of the ID complex. Thus, the mono-ubiquitylated ID complex recruits the downstream repair protein FAN1 and facilitates the repair of DNA interstrand cross-links.
Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells*♦

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Canver, Matthew C.; Bauer, Daniel E.; Dass, Abhishek; Yien, Yvette Y.; Chung, Jacky; Masuda, Takeshi; Maeda, Takahiro; Paw, Barry H.; Orkin, Stuart H.

2014-01-01

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements. PMID:24907273
Fen Eğitiminde Öğrenme Döngüsü Modelleri

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Yüksel Keleş

2011-11-01

Full Text Available Öğrenme döngüleri yapılandırıcı yaklaşımla fen eğitimi için geliştirilmiş modellerdir. Öğrenme döngüsü modelleri birbirini izleyen 3, 5 veya 7 evreden oluşmaktadır, bu evreler birbirinden ayrı ya da doğrusal değildir. Öğrenme döngüsü uygulamalarının yaratıcı ve eleştirel düşünme için öğrencileri teşvik etmesi ve bilime karşı olumlu tutum geliştirilmesi beklenmektedir. Kavramların daha iyi anlaşılmasına ve öğrencilerde ileri düşünme yeteneklerinin gelişmesine yardımcı olacağı düşünülmektedir. Bu makalede 5E ve 7E modellerinin her bir evresindeki öğrenci ve öğretmen rolleri üzerinde durulmaktadır. Model üzerinde gerçekleştirilen çalışmaların sonuçlarına dayanılarak modelin sağlayacağı olası faydalar tartışılmaktadır. Fen eğitiminde öğrenme döngüsü uygulamalarının yaygınlaştırılması amacıyla, öğretmen yetiştirme süreçlerinde öğrenme döngüsü modellerine yer verilmesi önerilmektedir.
CCR5 Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection

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Liu, Jingjing; Zhang, Di; Kimata, Jason T.; Zhou, Paul

2014-01-01

CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1. PMID:25541967
Partitioning of soil CO2 efflux in un-manipulated and experimentally flooded plots of a temperate fen

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Wunderlich, S.; Borken, W.

2012-08-01

Peatlands store large amounts of organic carbon, but the carbon stock is sensitive to changes in precipitation or water table manipulations. Restoration of drained peatlands by drain blocking and flooding is a common measure to conserve and augment the carbon stock of peatland soils. Here, we report to what extent flooding affected the contribution of heterotrophic and rhizosphere respiration to soil CO2 efflux in a grass-dominated mountain fen in Germany. Soil CO2 efflux was measured in three un-manipulated control plots and three flooded plots in two consecutive years. Flooding was achieved by permanent irrigation during the growing seasons. Radiocarbon signatures of CO2 from different sources including soil CO2 efflux, incubated peat cores and live grass roots were repeatedly analyzed for partitioning of soil CO2 efflux. Additionally, heterotrophic respiration and its radiocarbon signature were determined by eliminating rhizosphere respiration in trenched subplots (only control). In the control plots, rhizosphere respiration determined by 14C signatures contributed between 47 and 61% during the growing season, but was small (4 ± 8%) immediately before budding. Trenching revealed a smaller rhizosphere contribution of 33 ± 8% (2009) and 22 ± 9% (2010) during growing seasons. Flooding reduced annual soil CO2 efflux of the fen by 42% in 2009 and by 30% in 2010. The reduction was smaller in 2010 mainly through naturally elevated water level in the control plots. A one-week interruption of irrigation caused a strong short-lived increase in soil CO2 efflux, demonstrating the sensitivity of the fen to water table drawdown near the peat surface. The reduction in soil CO2 efflux in the flooded plots diminished the relative proportion of rhizosphere respiration from 56 to 46%, suggesting that rhizosphere respiration was slightly more sensitive to flooding than heterotrophic respiration.

Cell-based delivery of glucagon-like peptide-1 using encapsulated mesenchymal stem cells.

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Wallrapp, Christine; Thoenes, Eric; Thürmer, Frank; Jork, Anette; Kassem, Moustapha; Geigle, Peter

2013-01-01

Glucagon-like peptide-1 (GLP-1) CellBeads are cell-based implants for the sustained local delivery of bioactive factors. They consist of GLP-1 secreting mesenchymal stem cells encapsulated in a spherically shaped immuno-isolating alginate matrix. A highly standardized and reproducible encapsulation method is described for the manufacturing of homogeneous CellBeads. Viability and sustained secretion was shown for the recombinant GLP-1 and the cell endogenous bioactive factors like vascular endothelial growth factor, neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor. Manufacturing and quality control is performed in compliance with good manufacturing practice and fulfils all regulatory requirements for human clinical use. GLP-1 CellBeads combine the neuro- and cardioprotective properties of both GLP-1 and mesenchymal stem cells. First promising results were obtained from preclinical studies and an ongoing safety trial in humans but further studies have to prove the overall potential of CellBead technology in cell-based regenerative medicine.
A Mismatch EndoNuclease Array-Based Methodology (MENA) for Identifying Known SNPs or Novel Point Mutations.

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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.
Migração internacional de trabalho qualificado e o fenômeno do brain drain no Brasil

OpenAIRE

FARIA, Bruna Maia de

2008-01-01

Maia de Faria, Bruna; Barrantes Hidalgo, Álvaro. Migração internacional de trabalho qualificado e o fenômeno do brain drain no Brasil. 2008. Dissertação (Mestrado). Programa de Pós-Graduação em Economia, Universidade Federal de Pernambuco, Recife, 2008.
Using multi-proxy palaeoecology to test a relict status of refugial populations of calcareous-fen species in the Western Carpathians

Czech Academy of Sciences Publication Activity Database

Hájková, Petra; Horsák, M.; Hájek, Michal; Jankovská, Vlasta; Jamrichová, Eva; Moutelíková, J.

2015-01-01

Roč. 25, č. 4 (2015), s. 702-715 ISSN 0959-6836 Institutional support: RVO:67985939 Keywords : biodiversity * calcareous fen * fossil record * Holocene extinction * Western Carpathians Subject RIV: EF - Botanics Impact factor: 2.135, year: 2015
Water stress index for alkaline fen habitat based on UAV and continuous tower measurements of canopy infrared temperature

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Ciężkowski, Wojciech; Jóźwiak, Jacek; Chormański, Jarosław; Szporak-Wasilewska, Sylwia; Kleniewska, Małgorzata

2017-04-01

This study is focused on developing water stress index for alkaline fen, to evaluate water stress impact on habitat protected within Natura 2000 network: alkaline fens (habitat code:7230). It is calculated based on continuous measurements of air temperature, relative humidity and canopy temperature from meteorological tower and several UAV flights for canopy temperature registration. Measurements were taken during the growing season in 2016 in the Upper Biebrza Basin in north-east Poland. Firstly methodology of the crop water stress index (CWSI) determination was used to obtained non-water stress base line based on continuous measurements (NWSBtower). Parameters of NWSBtower were directly used to calculate spatial variability of CWSI for UAV thermal infrared (TIR) images. Then for each UAV flight day at least 3 acquisition were performed to define NWSBUAV. NWSBUAV was used to calculate canopy waters stress for whole image relative to the less stressed areas. The spatial distribution of developed index was verified using remotely sensed indices of vegetation health. Results showed that in analysed area covered by sedge-moss vegetation NWSB cannot be used directly. The proposed modification of CWSI allows identifying water stress in alkaline fen habitats and was called as Sedge-Moss Water Stress Index (SMWSI). The study shows possibility of usage remotely sensed canopy temperature data to detect areas exposed to the water stress on wetlands. This research has been carried out under the Biostrateg Programme of the Polish National Centre for Research and Development (NCBiR), project No.: DZP/BIOSTRATEG-II/390/2015: The innovative approach supporting monitoring of non-forest Natura 2000 habitats, using remote sensing methods (HabitARS).
Quantificação de compostos fenólicos e ácido ascórbico em frutos e polpas congeladas de acerola, caju, goiaba e morango

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Juliana Mesquita Freire

2013-12-01

Full Text Available O crescente interesse da população pela promoção da saúde vem despertando a atenção dos consumidores por alimentos ricos em antioxidantes, combinado com a praticidade do consumo de sucos de frutos processados. O presente trabalho objetivou quantificar e avaliar a capacidade antioxidante de compostos fenólicos e ácido ascórbico em frutas in natura e polpas congeladas. As frutas in natura e polpas congeladas obtidas na região de Lavras - MG foram submetidas à quantificação de compostos fenólicos e ácido ascórbico e os respectivos extratos acetônico-etanólico e acetônico-metanólico. Foram determinadas suas atividades antioxidantes pelos métodos radicalares DPPH e ABTS. O extrato acetônico-metanólico foi mais efetivo para extrair os compostos antioxidantes das amostras. Apenas as polpas congeladas de acerola apresentaram uma redução do teor de compostos fenólicos em relação ao fruto. Para o teor de ácido ascórbico, houve uma redução nas polpas congeladas de acerola e goiaba. As amostras de acerola apresentaram os maiores teores de compostos fenólicos, ácido ascórbico e atividade antioxidante em relação aos demais frutos analisados.
Direct stacking of sequence-specific nuclease-induced mutations to produce high oleic and low linolenic soybean oil.

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Demorest, Zachary L; Coffman, Andrew; Baltes, Nicholas J; Stoddard, Thomas J; Clasen, Benjamin M; Luo, Song; Retterath, Adam; Yabandith, Ann; Gamo, Maria Elena; Bissen, Jeff; Mathis, Luc; Voytas, Daniel F; Zhang, Feng

2016-10-13

The ability to modulate levels of individual fatty acids within soybean oil has potential to increase shelf-life and frying stability and to improve nutritional characteristics. Commodity soybean oil contains high levels of polyunsaturated linoleic and linolenic acid, which contribute to oxidative instability - a problem that has been addressed through partial hydrogenation. However, partial hydrogenation increases levels of trans-fatty acids, which have been associated with cardiovascular disease. Previously, we generated soybean lines with knockout mutations within fatty acid desaturase 2-1A (FAD2-1A) and FAD2-1B genes, resulting in oil with increased levels of monounsaturated oleic acid (18:1) and decreased levels of linoleic (18:2) and linolenic acid (18:3). Here, we stack mutations within FAD2-1A and FAD2-1B with mutations in fatty acid desaturase 3A (FAD3A) to further decrease levels of linolenic acid. Mutations were introduced into FAD3A by directly delivering TALENs into fad2-1a fad2-1b soybean plants. Oil from fad2-1a fad2-1b fad3a plants had significantly lower levels of linolenic acid (2.5 %), as compared to fad2-1a fad2-1b plants (4.7 %). Furthermore, oil had significantly lower levels of linoleic acid (2.7 % compared to 5.1 %) and significantly higher levels of oleic acid (82.2 % compared to 77.5 %). Transgene-free fad2-1a fad2-1b fad3a soybean lines were identified. The methods presented here provide an efficient means for using sequence-specific nucleases to stack quality traits in soybean. The resulting product comprised oleic acid levels above 80 % and linoleic and linolenic acid levels below 3 %.
Comparison of TALEN scaffolds in Xenopus tropicalis

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Keisuke Nakajima

2013-11-01

Transcription activator-like effector nucleases (TALENs are facile and potent tools used to modify a gene of interest for targeted gene knockout. TALENs consist of an N-terminal domain, a DNA-binding domain, and a C-terminal domain, which are derived from a transcription activator-like effector, and the non-specific nuclease domain of FokI. Using Xenopus tropicalis (X. tropicalis, we compared the toxicities and somatic mutation activities of four TALEN architectures in a side-by-side manner: a basic TALEN, a scaffold with the same truncated N- and C-terminal domains as GoldyTALEN, a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain, and a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric Sharkey nuclease domain. The strongest phenotype and targeted somatic gene mutation were induced by the injection of TALEN mRNAs containing the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain. The obligate heterodimeric TALENs exhibited reduced toxicity compared to the homodimeric TALENs, and the homodimeric GoldyTALEN-type scaffold showed both a high activity of somatic gene modification and high toxicity. The Sharkey mutation in the heterodimeric nuclease domain reduced the TALEN-mediated somatic mutagenesis.
Substâncias fenólicas, flavonoides e capacidade antioxidante em erveiras sob diferentes coberturas do solo e sombreamentos

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T.S. FERRERA

Full Text Available RESUMO Objetivou neste trabalho analisar a concentração de polifenóis totais, flavonoides e capacidade antioxidantes por meio dos métodos ABTS e FRAP, em erveiras jovens cultivadas em solo coberto e com sombreamento. Mudas de erva-mate foram submetidas aos tratamentos com solos cobertos e desnudos em sombreamentos de 0, 18, 35 e 50%, após sete e onze meses de cultivo, verão e outono, respectivamente. Foram coletadas folhas maduras para realização dos extratos utilizados para as análises de compostos fenólicos por meio da reação de oxirredução com reagente de Folin-Ciocalteu, flavonoides por método colorimétrico e capacidade antioxidante por frente ao radical ABTS e poder de redução do ferro (FRAP. O teor de compostos fenólicos foi maior nos tratamentos com 35% de sombreamento em comparação ao grupo dos flavonoides e capacidade antioxidante, o que foi demonstrado pela correlação do sombreamento com estes parametros. No método FRAP a maior correlação demonstra que o outono é a melhor época de colheita por apresentar maior concentração de compostos fitoquímicos. Ficou evidente a influência positiva dos sombreamentos (35 e 50% no teor de compostos fenólicos e flavonoides relacionados à capacidade antioxidante e à qualidade da erva-mate para atender ao mercado consumidor.
Atividade antimicrobiana, antioxidante e teor de compostos fenólicos em casca e amêndoa de frutos de manga

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Kettelin Aparecida Arbos

2013-04-01

Full Text Available O processamento industrial de frutos de manga gera elevada quantidade de resíduos agroindustriais, representados pelas cascas e sementes (amêndoa, os quais, sem aplicação viável, acabam sendo descartados diretamente no meio ambiente. Esses resíduos são ricos em compostos bioativos, amplamente reconhecidos pelas suas propriedades promotoras da saúde e em aplicações tecnológicas. Os objetivos foram avaliar as atividades antimicrobiana e antioxidante e o teor de compostos fenólicos de dois extratos: um obtido da casca (FC e outro da amêndoa (FA de manga variedade 'Tommy Atkins'. O teor de compostos fenólicos totais variou de 3.123 a 6.644 mg de catequina/100 g. Os extratos FC e FA demonstraram relevante atividade antimicrobiana frente às cepas das bactérias Escherichia coli, Salmonella sp., Pseudomonas aeruginosa . Staphylococcus aureus, determinadas pelo método de difusão em disco. A ação antioxidante de FC e FA aumentou com o aumento das concentrações testadas, atingindo o valor máximo de 88% (FC. Em todas as concentrações testadas os extratos FC apresentaram ação antioxidante significativamente superior às respectivas concentrações dos extratos FA. Esses resultados sugerem potencial aplicação dos resíduos de manga como fonte de compostos fenólicos, substâncias antimicrobianas e antioxidantes, podendo ser explorados pelas indústrias de alimentos.
14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

DEFF Research Database (Denmark)

Andersen, Sofie Dabros; Keijzers, Guido; Rampakakis, Emmanouil

2012-01-01

Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins...... are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding...... and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate...
Feminización del fenómeno migratorio ecuatoriano

Directory of Open Access Journals (Sweden)

Jorge Moreno Egas

2009-12-01

Full Text Available La crítica situación económica y política del Ecuador en la última década del XX fue la coyuntura que agudizó el fenómeno migratorio presente desde mediados de ese siglo. América Latina, Norte América y en los últimos tiempos Europa, particularmente España e Italia son los lugares de destino de los ecuatorianos. Este hecho se caracteriza por ser amplio y diverso. Los migrantes provienen de todas las clases sociales, es multigeneracional y particularmente femenino. Los efectos de la crisis y situaciones particulares de violencia y marginación han obligado a las mujeres a buscar alternativas de trabajo fuera del país. La feminización del fenómeno migratorio, dentro o fuera de la legalidad, más allá de su lado bueno, - remesas para la familia que queda a tras, éxitos personales en los lugares de acogida y la perspectiva de reunificación de la familia -, coloca a las migrantes en situaciones de vulnerabilidad, origina rupturas familiares y pone en riesgo la seguridad de los niños por privárseles del papel protagónico que las madres cumplen en su crianza.___________________ABSTRACT:Ecuador’s critical economic and political situation during the last decade of the Twentieth Century was the trend that worsened the emigration phenomenon present from the middle of the century. Latin America, North America and, lately, Europe, particularly Spain and Italy, are the destinations for Ecuadorians. This fact is characterized by being broad and diverse. Emigrants come from all social classes, are multi-generational and particularly female. The effects of the crisis and the specific situations of violence and marginalization have forced women to seek options for work outside the country. The feminization of the emigration phenomenon inside or outside the law other than its good side – remittances to the family that stayed behind, personal success in the places where they are received and the perspective of reuniting with family �
Potencial de actividad antioxidante de extractos fenólicos de Theobroma cacao L. (cacao)

OpenAIRE

Quiñones Gálvez, Janet; Trujillo Sánchez, Reinaldo; Capdesuñer Ruiz, Yanelis; Quirós Molina, Yemeys; Hernández de la Torre, Martha

2013-01-01

Introducción: Theobroma cacao L. (cacao), familia Sterculiaceae, es una planta rica en fenoles, los cuales en las plantas son de especial interés por su potente actividad antioxidante, dada esencialmente por sus propiedades redox, que pueden desempeñar un papel importante en la absorción y neutralización de los radicales libres. Objetivos: seleccionar un clon promisorio en la producción de compuestos fenólicos, determinar los órganos con mayor contenido de fenoles, establecer el cultivo in vi...
Long Noncoding RNA lncCAMTA1 Promotes Proliferation and Cancer Stem Cell-Like Properties of Liver Cancer by Inhibiting CAMTA1

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Li-Juan Ding

2016-09-01

Full Text Available Hepatocellular carcinoma (HCC is the most common subtype of liver malignancy, and it is characterized by poor prognosis because of cancer stem cell (CSC-mediated high postsurgical recurrence rates. Thus, targeting CSCs, or HCC cells with CSC-like properties, is an effective strategy for HCC therapy. Here, using long noncoding RNA (lncRNA microarray analysis, we identified a novel lncRNA termed lncCAMTA1 that is increased in both liver CSCs and HCC. High lncCAMTA1 expression in HCC indicates poor clinical outcome. In vitro and in vivo functional experiments showed that overexpression of lncCAMTA1 promotes HCC cell proliferation, CSC-like properties, and tumorigenesis. Conversely, depletion of lncCAMTA1 inhibits HCC cell proliferation, CSC-like properties, and tumorigenesis. Mechanistically, we demonstrated that lncCAMTA1 physically associates with the calmodulin binding transcription activator 1 (CAMTA1 promoter, induces a repressive chromatin structure, and inhibits CAMTA1 transcription. Furthermore, CAMTA1 is required for the effects of lncCAMTA1 on HCC cell proliferation and CSC-like properties, and the expression of lncCAMTA1 and CAMTA1 is significantly negatively correlated in HCC tissues. Collectively, our study revealed the important roles and underlying molecular mechanisms of lncCAMTA1 on HCC, and suggested that lncCAMTA1 could be an effective prognostic factor and a potential therapeutic target for HCC.
El fenómeno del apoyo al aprendizaje fuera de la escuela visto a través de un análisis comparado

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Alexandre Ventura

2006-01-01

Full Text Available La prácticas de apoyo al aprendizaje fuera de la escuela, que se analizan en este artículo, han merecido nuestra atención por diversas razones: 1 el hecho de que son prácticamente un fenómeno mundial, a pesar de los sistemas educativos difieran de un país a otro; 2 el hecho de que las repercusiones de las explicaciones sobre los resultados escolares puedan llevar a una reubicación de los principios de equidad e igualdad de oportunidades; 3 la constatación de que los costes financieros soportados por las familias de los alumnos, por un lado, y el número creciente de explicadores disponibles (trabajando a tiempo parcial o en jornada completa, por otro, confieren a esta actividad una importancia social y económica considerable. Este abordaje prima un análisis cualitativo del fenómeno de las clases particulares, con el objetivo de conocer mejor algunas de sus formas: su ejercicio en exclusividad o como complemento del salario obtenido en el decurso de otra actividad; como iniciativa de base individual, característica de una actividad liberal; en el contexto de un centro/academia local; o en el contexto de un centro de estudios con franquicia, de dimensión nacional o multinacional. Al realizar un análisis comparado de este fenómeno, buscaremos mostrar como las explicaciones constituyen una actividad promovida de forma creciente en organizaciones (centros de explicaciones, sobre todo en los grandes centros urbanos. Estos centros de explicaciones están para suplementar o competir con las escuelas utilizando metodologías de sistematización o especialización.
Leaf-litter microfungal community on poor fen plant debris in Torfy Lake area (Central Poland)

OpenAIRE

Mateusz Wilk; Agnieszka Banach; Julia Pawłowska; Marta Wrzosek

2014-01-01

The purpose of this study was to initially evaluate the species diversity of microfungi growing on litter of 15 plant species occurring on the poor fen and neighbouring area of the Torfy Lake, Masovian voivodeship, Poland. The lake is located near the planned road investment (construction of the Warsaw southern express ring road S2). The place is biologically valuable as there are rare plant communities from Rhynchosporion albae alliance protected under the Habitats Directive adopted by the E...
Efeito de diferentes métodos de extração sobre a atividade antioxidante e o perfil de compostos fenólicos da folha de mandioca

Directory of Open Access Journals (Sweden)

Mírian Aparecida Isidro Santos

Full Text Available Resumo Folhas de mandioca (Manihot esculenta Crantz são resíduos agroindustriais que apresentam potencial para o consumo humano, sendo fontes de proteínas, vitaminas, minerais e de substâncias antioxidantes, com destaque para os compostos fenólicos. No presente estudo, o objetivo foi identificar e quantificar os compostos fenólicos presentes em diferentes extratos da farinha de folhas de mandioca (FFM, cultivar Pão da China, e avaliar seu potencial antioxidante. A extração dos compostos fenólicos foi realizada utilizando três métodos de extração: a refluxo com metanol/água (50:50, v/v; b maceração com etanol/água (50:50, v/v seguido de refluxo com acetona/água (70:30, v/v; c maceração com metanol PA. A caracterização e quantificação foi realizada por cromatografia líquida de alta eficiência e a atividade antioxidante, determinada pelos métodos ABTS e β-caroteno/ácido linoleico. Foram encontrados cinco compostos fenólicos nos extratos da FFM: ácido gálico, galocatequina, catequina, ácido clorogênico e epigalocatequina, sendo a extração com metanol/água (50:50, v/v a que revelou a presença do maior número destes compostos e o maior potencial antioxidante. Entre os fenólicos da FFM, independentemente do extrato, a catequina foi o majoritário. Pode-se inferir que a atividade antioxidante dos extratos da FFM está relacionada com a presença de compostos, pois a extração com maior teor desses compostos foi a que demonstrou maior potencial antioxidante.
Compuestos fenólicos del olivo (Olea europea

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Maestro-Durán, R.

1994-08-01

Full Text Available Certain plant properties, such as the colour, the nutritional value, the resistance to microorganisms, the pharmacologic effects and the organoleptic character, are due to the phenolic compounds. It is interesting the study of phenolic compounds from olive because they:
-Inhibit Gram+ microorganisms, such as Lactobacillus, which are involved in the fermentation of the olive fruit. -Inhibit the activity of cellulases and other enzymes. -Contribute to the stability and organoleptic character of virgin olive oil. -Inhibit the effect of microorganisms involved in the anaerobic depuration of vegetation water. Thus, the phenolic compounds are the major bothersome contaminants of these wastewater. -Have pharmacologic properties (to reduce blood pressure levels. -Are natural antioxidants.
In the present review we describe the composition of the phenolic compounds from the olive fruit (pulp and seed, the olive oil and its by-products (vegetation water and rape and the olive leaves.

Ciertas propiedades de las plantas, como el color, valor nutricional, autoprotección contra microorganismos, propiedades farmacológicas o caracteres organolépticos, son debidas a los compuestos fenólicos. Su estudio, en el caso del olivo, es de gran interés por diversas razones:
-Por su carácter antibiótico, los fenoles de la aceituna inhiben los microorganismos Gram+, como Lactobacillus, necesarios para la fermentación de aceitunas de mesa. -Inhiben la acción de las celulasas y otras enzimas. -Son los responsables de la alta estabilidad de los aceites vírgenes de oliva, así como de alguno de sus caracteres organolépticos. -Inhiben la acción de los microorganismos responsables de la depuración anaerobia de los alpechines y son las sustancias contaminantes de estas aguas residuales cuya degradación para eliminar la DQO presenta más dificultades. -Tienen propiedades
Fatores implicados no fenómeno de bullying em contexto escolar: revisão integrada da literatura Factores implicados en el fenómeno de acoso en contexto escolar: revisión integrada de la literatura Factors related to the bullying phenomenon in school context: integrative literature review

Directory of Open Access Journals (Sweden)

Rita Lopez

2011-12-01

Full Text Available As evidências científicas sugerem que o bullying é um fenómeno cada vez mais frequente no contexto escolar, ao qual está inerente um vasto número de consequências para a saúde, pelo que, a sua prevenção deve constituir uma área prioritária. Com o objetivo de identificar os fatores implicados no fenómeno do bullying em contexto escolar, efetuou-se uma revisão da literatura de forma sistematizada na Biblioteca do Conhecimento Online (b-on com as seguintes palavras-chave: “bullying/bullying school context”, considerando os seguintes critérios de inclusão: participantes (estudantes do 2º e 3º ciclo; variáveis em estudo (fatores implicados no fenómeno do bullying, sem restrições relativas ao desenho dos estudos. Foram identificados 351 artigos, dos quais não foi possível aceder a 132. Considerando os critérios de inclusão, foram eliminados 203 artigos, os restantes 16 foram analisados. Da análise dos resultados dos estudos que integram esta revisão da literatura, emergem quatro tipologias de fatores, os fatores relacionados com as variáveis sociodemográficas, os fatores relacionados com as variáveis pessoais, os relativos à dimensão familiar e ainda os relacionados com as variáveis escolares. Em função destas tipologias, identificaram-se diversos fatores protetores e fatores de risco implicados no fenómeno de bullying.Las evidencias científicas sugieren que el acoso es un fenómeno cada vez más frecuente en el contexto escolar, al cual está inherente un vasto número de consecuencias para la salud, por lo que su prevención debe constituir un área prioritaria. Con el objetivo de identificar a los factores implicados en el fenómeno del acoso en contexto escolar, se efectuó una revisión de la literatura de forma sistematizada en la “Biblioteca do Conhecimento Online” (b-on con las siguientes palabras clave: “bullying/bullying school context”, considerando los siguientes criterios de inclusi
Mixed ligand complexes of Cu(II)/Zn(II) ions containing (m-)/(p-) carboxylato phenyl azo pentane 2,4-dione and 2,2'-bipyridine/1,10 phenanthroline: Synthesis, characterization, DNA binding, nuclease and topoisomerase I inhibitory activity.

Science.gov (United States)

Hasan, Md Amin; Kumari, Niraj; Singh, Kanhaiya; Singh, Kiran; Mishra, Lallan

2016-01-05

Metal complexes of type [Cu(L1H)2(bpy)] (1), [Zn(L1H)2(bpy)] (2), [Cu(L2H)2(bpy)] (3) and [Cu(L2H)2(Phen)] (4) (L1H2=3-[N'-(1-acetyl-2-oxo-propylidene)-hydrazino]-benzoic acid, L2H2=4-[N'-(1-acetyl-2-oxo-propylidene)-hydrazino]-benzoic acid, bpy=2,2'-bipyridine, Phen=1,10 phenanthroline) are synthesized and characterized using spectroscopic techniques (FT-IR, (1)H NMR, (13)C NMR, electronic absorption and emission) and elemental analysis data. The assembly of the complexes involving intramolecular H-bonding is displayed using corresponding crystal structure. Binding of the complexes separately with Calf Thymus DNA is monitored using UV-vis spectral titrations. The displacement of ethidium bromide (EB) bound to DNA by the complexes, in phosphate buffer solution (pH∼7.2) is monitored using fluorescence spectral titrations. Nuclease activity of the complexes follow the order 4>3>1>2. The gel electrophoretic mobility assay measurement in presence of minor groove binder 4',6-diamidino-2-phenylindole (DAPI), suggests that complexes preferably bind with the minor groove of DNA. Topoisomerase I inhibitory activity of the complexes 3 and 4 inhibit topoisomerase I activity with IC50 values of 112 and 87μM respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

Mobile Genetic Elements and Evolution of CRISPR-Cas Systems: All the Way There and Back

Science.gov (United States)

Makarova, Kira S.

2017-01-01

Abstract The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) systems of bacterial and archaeal adaptive immunity show multifaceted evolutionary relationships with at least five classes of mobile genetic elements (MGE). First, the adaptation module of CRISPR-Cas that is responsible for the formation of the immune memory apparently evolved from a Casposon, a self-synthesizing transposon that employs the Cas1 protein as the integrase and might have brought additional cas genes to the emerging immunity loci. Second, a large subset of type III CRISPR-Cas systems recruited a reverse transcriptase from a Group II intron, providing for spacer acquisition from RNA. Third, effector nucleases of Class 2 CRISPR-Cas systems that are responsible for the recognition and cleavage of the target DNA were derived from transposon-encoded TnpB nucleases, most likely, on several independent occasions. Fourth, accessory nucleases in some variants of types I and III toxin and type VI effectors RNases appear to be ultimately derived from toxin nucleases of microbial toxin–antitoxin modules. Fifth, the opposite direction of evolution is manifested in the recruitment of CRISPR-Cas systems by a distinct family of Tn7-like transposons that probably exploit the capacity of CRISPR-Cas to recognize unique DNA sites to facilitate transposition as well as by bacteriophages that employ them to cope with host defense. Additionally, individual Cas proteins, such as the Cas4 nuclease, were recruited by bacteriophages and transposons. The two-sided evolutionary connection between CRISPR-Cas and MGE fits the “guns for hire” paradigm whereby homologous enzymatic machineries, in particular nucleases, are shuttled between MGE and defense systems and are used alternately as means of offense or defense. PMID:28985291
Human placental growth hormone, insulin-like growth factor I and -II, and insulin requirements during pregnancy in type 1 diabetes

DEFF Research Database (Denmark)

Fuglsang, Jens; Lauszus, Finn; Flyvbjerg, Allan

2003-01-01

between hPGH and IGF-I in type 1 diabetes mellitus has not been investigated thoroughly. Furthermore, hPGH may be involved in the development of insulin resistance during pregnancy. In this prospective, longitudinal study, 51 type 1 diabetic subjects were followed with repeated blood sampling during......PGH was not correlated to the increase in insulin requirements, nor was any consistent relationship revealed during each gestational period. In conclusion, our study suggests a role for hPGH in the regulation of both IGFs and fetal growth in type 1 diabetes. In contrast, the increase in insulin requirements during...... pregnancy in type 1 diabetic subjects could not be related to hPGH levels....
ORF Alignment: NC_000913 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available junction ... nuclease; resolution of structures; repair; Holliday ... junction nuclease [Esche...richia coli K12] gb|AAG56853.1| ... Holliday junction nuclease; resolution of structures...|NP_288300.1| Holliday junction nuclease; ... resolution of structures; repair [Escherichia coli ...
ORF Alignment: NC_002695 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available junction ... nuclease; resolution of structures; repair; Holliday ... junction nuclease [Esche...richia coli K12] gb|AAG56853.1| ... Holliday junction nuclease; resolution of structures...|NP_288300.1| Holliday junction nuclease; ... resolution of structures; repair [Escherichia coli ...
ORF Alignment: NC_004431 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available junction ... nuclease; resolution of structures; repair; Holliday ... junction nuclease [Esche...richia coli K12] gb|AAG56853.1| ... Holliday junction nuclease; resolution of structures...|NP_288300.1| Holliday junction nuclease; ... resolution of structures; repair [Escherichia coli ...
ORF Alignment: NC_002655 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available junction ... nuclease; resolution of structures; repair; Holliday ... junction nuclease [Esche...richia coli K12] gb|AAG56853.1| ... Holliday junction nuclease; resolution of structures...|NP_288300.1| Holliday junction nuclease; ... resolution of structures; repair [Escherichia coli ...
Lucio's phenomenon: another case reported in Brazil Fenômeno de Lúcio: mais um caso relatado no Brazil

Directory of Open Access Journals (Sweden)

Rodrigo Monteiro

2012-04-01

Full Text Available Lucio's phenomenon is defined as a variant of type 2 leprosy reaction. It is a rare event, occurring in the evolution of leprosy of Lucio and other forms of lepromatous leprosy. It has an exacerbated proliferation of Hansen bacilli in its pathophysiology, which invade blood vessel walls and injure endothelial cells, causing endothelial proliferation and decreasing the vascular lumen. This fact, associated with inflammatory reactions and changes in the coagulation system causes vascular thrombosis, ischemia, infarction and tissue necrosis, leading to the histopathological characteristic of the phenomenon. We report a case of lepromatous leprosy with irregular treatment that developed Lucio's phenomenon. Treatment with multidrug therapy, antibiotics, steroids and thalidomide achieved a favorable outcome.Define-se o fenômeno de Lúcio como uma variante da reação hansênica do tipo 2. Evento raro, que ocorre na evolução da hanseníase de Lúcio e de outras formas de hanseníase virchowiana. Tem na sua fisiopatologia uma proliferação exacerbada dos bacilos de Hansen, que invadem a parede dos vasos sanguíneos e agridem as células endoteliais, causando proliferação endotelial e diminuição do lúmen vascular, fato este, que associado a reações inflamatórias e a alterações no sistema da coagulação, causa trombose vascular, isquemia, infarto e necrose tecidual, gerando as alterações histopatológicas características do fenômeno. Relatamos um caso de hanseníase virchowiana, com tratamento irregular, que desenvolveu o fenômeno de Lúcio. Recebeu tratamento com poliquimioterapia, antibióticos, corticosteróide e talidomida, evoluindo com desfecho clínico favorável.
Fe-N and (Fe, Ni)-N Fine Powders for Magnetic Recording

International Nuclear Information System (INIS)

Chen Ziyu; Li Fashen

1998-01-01

Combining Moessbauer spectroscopy with magnetic property measurement, we have studied Fe-N and (Fe, Ni)-N powders for magnetic recording. The typical particles of the core (α-Fe)/shell (γ'-Fe 4 N) structure have been successfully prepared. All the products are stabilized in a multi-organic solution. It has been found that the coercivity can be changed from 300 to 800 Oe by adjusting the shape of the particles. The special saturation magnetization of the particles can be adjusted from 120 to 180emu/g and their chemical stability is improved by substituting nickel for iron in γ'-Fe 4 N. Following experiments for corrosion resistance, it is expected that (Fe, Ni)-N and the core/shell particles will be applied as recording media in the near future
Fe-N and (Fe, Ni)-N Fine Powders for Magnetic Recording

Energy Technology Data Exchange (ETDEWEB)

Chen Ziyu; Li Fashen [Lanzhou University, Department of Physics (China)

1998-12-15

Combining Moessbauer spectroscopy with magnetic property measurement, we have studied Fe-N and (Fe, Ni)-N powders for magnetic recording. The typical particles of the core ({alpha}-Fe)/shell ({gamma}'-Fe{sub 4}N) structure have been successfully prepared. All the products are stabilized in a multi-organic solution. It has been found that the coercivity can be changed from 300 to 800 Oe by adjusting the shape of the particles. The special saturation magnetization of the particles can be adjusted from 120 to 180emu/g and their chemical stability is improved by substituting nickel for iron in {gamma}'-Fe{sub 4}N. Following experiments for corrosion resistance, it is expected that (Fe, Ni)-N and the core/shell particles will be applied as recording media in the near future.
Heidegger: o estudo dos fenômenos humanos baseados na existência humana como ser-aí (Dasein

Directory of Open Access Journals (Sweden)

Ida Elizabeth Cardinalli

2015-08-01

Full Text Available O presente artigo pretende esclarecer a posição crítica do filósofo Martin Heidegger, explicitada no livro Seminários de Zollikon, em relação à transposição do método da ciência natural moderna para o estudo dos fenômenos humanos e apresentar sua proposição de um método mais pertinente ao estudo da existência humana. O autor propõe indicações de um método baseado na compreensão do existir humano como "ser-aí" e "ser-no-mundo" e também nos existenciais descritos em Ser e tempo, tais como espacialidade, temporalidade, ser-com-o-outro e corporeidade. Ao mesmo tempo, destaca que os fenômenos humanos não deveriam ser submetidos e circunscritos às descrições ontológicas dos existenciais, quando o método visa ao esclarecimento das experiências específicas e singulares de cada ser humano.
Phosphate release upon short- and long-term flooding of fen meadows depends on land use history and pH.

NARCIS (Netherlands)

Beltman, Boudewijn; van der Ven, P.J.M.; Verhoeven, Jos; Sarneel, J

2014-01-01

Flooding of acidified and desiccated fen meadows is a management approach for mitigating loss of plant species as well as a short-term measure to prevent flooding in urban areas. Studies have shown that flooding events can cause extreme P release from soils. We questioned whether the occurrence of
Sobre los saberes culturales y el fenómeno "sentirse bien-sentirse mal" On cultural knowledge and the phenomenon "feel good-fell bad"

OpenAIRE

Virtudes Rodero Sánchez; Carme Vives Relats; Sagrario Acebedo Urdiales; Carme Ferré Grau

2010-01-01

El objetivo del presente estudio es identificar los saberes culturales inmersos en el fenómeno "sentirse-bien, sentirse mal", en diferentes situaciones de cuidado. Se elaboró un estudio cualitativo con un enfoque fenomenológico interpretativo, mediante el análisis de 18 narrativas a partir de entrevistas en profundidad realizadas por alumnas del Máster en Ciencias de la Enfermería en diferentes contextos de cuidados. Las categorías identificadas fueron: (1) Cronicidad y concepto de sentirse b...
Fen Ve Teknoloji Dersi Öğretmenlerinin SBS’de Yapılan Değişikliklerle İlgili Görüşlerinin İncelenmesi

Directory of Open Access Journals (Sweden)

MUSTAFA UZOĞLU

2013-09-01

Full Text Available Bu çalışma fen ve teknoloji öğretmenlerinin SBS’nin kaldırılması hakkındaki görüşlerini belirlemek amacıyla yapılmıştır. Veri toplama aracı olarak açık uçlu anketten yararlanılmıştır. Çalışmanın örneklemini 2012-2013 öğretim yılında Giresun İlinde görev yapan 51 fen ve teknoloji öğretmeni oluşturmaktadır. Açık uçlu anket formundan elde edilen sonuçlarda çalışmaya katılan fen ve teknoloji öğretmenlerinin birçoğu söz konusu sınavın kaldırılmasına karşı çıkmışlardır. Ayrıca öğretmenler sınav kaldırıldığında öğrencilerin yerleştirilmesinde sıkıntıların çıkabileceğini ifade etmişlerdir. Diğer yandan fen ve teknoloji öğretmenlerinin ortaöğretim kurumlarına öğrenci seçmek için yapılan sınavların sayısının ve şeklinin sürekli değişmesinden dolayı endişelendikleri dikkati çekmektedir. Fen ve teknoloji öğretmenleri bu durumun öğrencilerde güven kaybına, motivasyon düşüklüğüne, karamsarlığa bıkkınlığa, gelecek endişesine, adaptasyon sorununa neden olabileceğini ifade etmişlerdir.
NM23-H2 may play an indirect role in transcriptional activation of c-myc gene expression but does not cleave the nuclease hypersensitive element III1

International Nuclear Information System (INIS)

Dexheimer, Thomas S.; Carey, Steven S.; Zuohe, Song; Gokhale, Vijay M.; Hu, Xiaohui; Murata, Lauren B.; Maes, Estelle M.; Weichsel, Andrzej; Sun, Daekyu; Meuillet, Emmanuelle J.; Montfort, William R.; Hurley, Laurence H.

2009-01-01

The formation of G-quadruplex structures within the nuclease hypersensitive element (NHE) III 1 region of the c-myc promoter and the ability of these structures to repress c-myc transcription have been well established. However, just how these extremely stable DNA secondary structures are transformed to activate c-myc transcription is still unknown. NM23-H2/nucleoside diphosphate kinase B has been recognized as an activator of c-myc transcription via interactions with the NHE III 1 region of the c-myc gene promoter. Through the use of RNA interference, we confirmed the transcriptional regulatory role of NM23-H2. In addition, we find that further purification of NM23-H2 results in loss of the previously identified DNA strand cleavage activity, but retention of its DNA binding activity. NM23-H2 binds to both single-stranded guanine- and cytosine-rich strands of the c-myc NHE III 1 and, to a lesser extent, to a random single-stranded DNA template. However, it does not bind to or cleave the NHE III 1 in duplex form. Significantly, potassium ions and compounds that stabilize the G-quadruplex and i-motif structures have an inhibitory effect on NM23-H2 DNA-binding activity. Mutation of Arg 88 to Ala 88 (R88A) reduced both DNA and nucleotide binding but had minimal effect on the NM23-H2 crystal structure. On the basis of these data and molecular modeling studies, we have proposed a stepwise trapping-out of the NHE III 1 region in a single-stranded form, thus allowing single-stranded transcription factors to bind and activate c-myc transcription. Furthermore, this model provides a rationale for how the stabilization of the G-quadruplex or i-motif structures formed within the c-myc gene promoter region can inhibit NM23-H2 from activating c-myc gene expression.
Spacer capture and integration by a type I-F Cas1-Cas2-3 CRISPR adaptation complex.

Science.gov (United States)

Fagerlund, Robert D; Wilkinson, Max E; Klykov, Oleg; Barendregt, Arjan; Pearce, F Grant; Kieper, Sebastian N; Maxwell, Howard W R; Capolupo, Angela; Heck, Albert J R; Krause, Kurt L; Bostina, Mihnea; Scheltema, Richard A; Staals, Raymond H J; Fineran, Peter C

2017-06-27

CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1-Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas1 4 -Cas2-3 2 complex from Pectobacterium atrosepticum with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1-Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1-Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3'-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1-Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1-Cas2-3 adaptation complexes and their integration mechanism.
Activation of the LRR Receptor-Like Kinase PSY1R Requires Transphosphorylation of Residues in the Activation Loop

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Christian B. Oehlenschlæger

2017-11-01

Full Text Available PSY1R is a leucine-rich repeat (LRR receptor-like kinase (RLK previously shown to act as receptor for the plant peptide hormone PSY1 (peptide containing sulfated tyrosine 1 and to regulate cell expansion. PSY1R phosphorylates and thereby regulates the activity of plasma membrane-localized H+-ATPases. While this mechanism has been studied in detail, little is known about how PSY1R itself is activated. Here we studied the activation mechanism of PSY1R. We show that full-length PSY1R interacts with members of the SERK co-receptor family in planta. We identified seven in vitro autophosphorylation sites on serine and threonine residues within the kinase domain of PSY1R using mass spectrometry. We furthermore show that PSY1R autophosphorylation occurs in trans and that the initial transphosphorylation takes place within the activation loop at residues Ser951, Thr959, and Thr963. While Thr959 and Thr963 are conserved among other related plant LRR RLKs, Ser951 is unique to PSY1R. Based on homology modeling we propose that phosphorylation of Ser951 stabilize the inactive conformation of PSY1R.
El fenómeno MOOC y la universalidad de la cultura: las nuevas fronteras de la educación superior

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Jose Gómez Galán

2014-01-01

Full Text Available Este artículo tiene como principal objetivo ofrecer una explicación a la rápida extensión del fenómeno MOOC –y su verdadera relevancia y posición en la Educación Superior– en el contexto de las dinámicas postmodernas de universalidad de la cultura, la comunicación y el conocimiento. Analiza, por medio de una metodología crítico-teórica y ex post facto, cómo los cursos MOOC representan una parte de la integración del mundo universitario en los complejos procesos de globalización propiciados por la revolución de las TIC, pero concluye que en modo alguno significan la última frontera de la Educación Superior. Como resultados más importantes se determina que contribuyen en cierta media a [1] una universalidad de la cultura, ubicándose dentro del fenómeno de la globalización y lo que hoy ésta representa, [2] la universalidad de la comunicación, en el seno de la convergencia tecno-mediática, [3] la universalidad del conocimiento, con el empleo de pedagogías y metodologías didácticas extendidas de modo global y, en definitiva, [4] la universalidad de la propia Educación Superior, partícipes de los procesos de homogenización de los modelos de estudios universitarios y de adaptación a las características de una sociedad en red. Pero no suponen un fenómeno de tal envergadura que lleve a una transformación radical de este nivel educativo. Ni siquiera ofrecen innovaciones pedagógicas y didácticas significativas en relación con lo ya aportado previa y paralelamente por otros procesos de e-learning. Significan tan sólo una de las muchas respuestas –en absoluto la única– a las nuevas necesidades sociales, en un contexto de formación para todos y durante toda la vida, lo que explica su eclosión.
Avr4 promotes Cf-4 receptor-like protein association with the BAK1/SERK3 receptor-like kinase to initiate receptor endocytosis and plant immunity.

Science.gov (United States)

Postma, Jelle; Liebrand, Thomas W H; Bi, Guozhi; Evrard, Alexandre; Bye, Ruby R; Mbengue, Malick; Kuhn, Hannah; Joosten, Matthieu H A J; Robatzek, Silke

2016-04-01

The first layer of plant immunity is activated by cell surface receptor-like kinases (RLKs) and proteins (RLPs) that detect infectious pathogens. Constitutive interaction with the SUPPRESSOR OF BIR1 (SOBIR1) RLK contributes to RLP stability and kinase activity. As RLK activation requires transphosphorylation with a second associated RLK, it remains elusive how RLPs initiate downstream signaling. We employed live-cell imaging, gene silencing and coimmunoprecipitation to investigate the requirement of associated kinases for functioning and ligand-induced subcellular trafficking of Cf RLPs that mediate immunity of tomato against Cladosporium fulvum. Our research shows that after elicitation with matching effector ligands Avr4 and Avr9, BRI1-ASSOCIATED KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3) associates with Cf-4 and Cf-9. BAK1/SERK3 is required for the effector-triggered hypersensitive response and resistance of tomato against C. fulvum. Furthermore, Cf-4 interacts with SOBIR1 at the plasma membrane and is recruited to late endosomes upon Avr4 trigger, also depending on BAK1/SERK3. These observations indicate that RLP-mediated resistance and endocytosis require ligand-induced recruitment of BAK1/SERK3, reminiscent of BAK1/SERK3 interaction and subcellular fate of the FLAGELLIN SENSING 2 (FLS2) RLK. This reveals that diverse classes of cell surface immune receptors share common requirements for initiation of resistance and endocytosis. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.
Dissociable Hippocampal and Amygdalar D1-like receptor contribution to Discriminated Pavlovian conditioned approach learning

Science.gov (United States)

Andrzejewski, Matthew E; Ryals, Curtis

2016-01-01

Pavlovian conditioning is an elementary form of reward-related behavioral adaptation. The mesolimbic dopamine system is widely considered to mediate critical aspects of reward-related learning. For example, initial acquisition of positively-reinforced operant behavior requires dopamine (DA) D1 receptor (D1R) activation in the basolateral amygdala (BLA), central nucleus of the amygdala (CeA), and the ventral subiculum (vSUB). However, the role of D1R activation in these areas on appetitive, non-drug-related, Pavlovian learning is not currently known. In separate experiments, microinfusions of the D1-like receptor antagonist SCH-23390 (3.0 nmol/0.5 μL per side) into the amygdala and subiculum preceded discriminated Pavlovian conditioned approach (dPCA) training sessions. D1-like antagonism in all three structures impaired the acquisition of discriminated approach, but had no effect on performance after conditioning was asymptotic. Moreover, dissociable effects of D1-like antagonism in the three structures on components of discriminated responding were obtained. Lastly, the lack of latent inhibition in drug-treated groups may elucidate the role of D1-like in reward-related Pavlovian conditioning. The present data suggest a role for the D1 receptors in the amygdala and hippocampus in learning the significance of conditional stimuli, but not in the expression of conditional responses. PMID:26632336
Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells.

Science.gov (United States)

Canver, Matthew C; Bauer, Daniel E; Dass, Abhishek; Yien, Yvette Y; Chung, Jacky; Masuda, Takeshi; Maeda, Takahiro; Paw, Barry H; Orkin, Stuart H

2014-08-01

The clustered regularly interspaced short [corrected] palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Mixed ligand complexes of Cu(II)/Zn(II) ions containing (m-)/(p-) carboxylato phenyl azo pentane 2,4-dione and 2,2‧-bipyridine/1,10 phenanthroline: Synthesis, characterization, DNA binding, nuclease and topoisomerase I inhibitory activity

Science.gov (United States)

Hasan, Md. Amin; Kumari, Niraj; Singh, Kanhaiya; Singh, Kiran; Mishra, Lallan

2016-01-01

Metal complexes of type [Cu(L1H)2(bpy)] (1), [Zn(L1H)2(bpy)] (2), [Cu(L2H)2(bpy)] (3) and [Cu(L2H)2(Phen)] (4) (L1H2 = 3-[N‧-(1-acetyl-2-oxo-propylidene)-hydrazino]-benzoic acid, L2H2 = 4-[N‧-(1-acetyl-2-oxo-propylidene)-hydrazino]-benzoic acid, bpy = 2,2‧-bipyridine, Phen = 1,10 phenanthroline) are synthesized and characterized using spectroscopic techniques (FT-IR, 1H NMR, 13C NMR, electronic absorption and emission) and elemental analysis data. The assembly of the complexes involving intramolecular H-bonding is displayed using corresponding crystal structure. Binding of the complexes separately with Calf Thymus DNA is monitored using UV-vis spectral titrations. The displacement of ethidium bromide (EB) bound to DNA by the complexes, in phosphate buffer solution (pH ∼ 7.2) is monitored using fluorescence spectral titrations. Nuclease activity of the complexes follow the order 4 > 3 > 1 > 2. The gel electrophoretic mobility assay measurement in presence of minor groove binder 4‧,6-diamidino-2-phenylindole (DAPI), suggests that complexes preferably bind with the minor groove of DNA. Topoisomerase I inhibitory activity of the complexes 3 and 4 inhibit topoisomerase I activity with IC50 values of 112 and 87 μM respectively.
Characterizing Early Succession Following Wildfires at Different Severities in Boreal Bog and Fen Peatlands

Science.gov (United States)

Ernst, E. J.; Bourgeau-Chavez, L. L.; Kane, E. S.; Wagenbrenner, J. W.; Endres, S.

2016-12-01

The Arctic-boreal region is experiencing changes in climate, trending toward warmer summers, resulting in a greater occurrence of wildfires with longer burning periods and higher intensities. Drought-like conditions have dried surface fuels, leading to a higher probability of ignition, even in lowland peatlands. Previous work has been done to characterize post-fire succession rates in Arctic-boreal upland sites, but much less is known of fire effects and early successional dynamics in lowlands. Wildland fires are the number one disturbance in Canada's Northwest Territories (NWT), which characteristically burn at high intensities with large flame fronts, and result in some of the biggest wildfires in the world. Areas surrounding the Great Slave Lake, NWT—including parts of the Taiga Plains, Taiga Shield, and Boreal Plains ecozones—experienced exceptional wildfire activity in 2014 and 2015. We characterized burn severity of the bog and fen peat surface and canopy layers at several burned sites. To determine if the severe ground or crown wildfires were stand-replacing events, we characterized post-fire vegetation in peatlands in 2015 and 2016 based on seedling regeneration. We stratified sites according to estimated water residence times across the three ecozones and made comparisons between data collected at the same sites across years. This work adds much needed context for post-fire succession in boreal peatland ecosystems, as the susceptibility of these systems to burning will continue to increase with a warming climate.
Fenólicos totais e atividade antioxidante do extrato aquoso de broto de feijão-mungo (Vigna radiata L. Total phenolics and antioxidant activity of the aqueous extract of mung bean sprout (Vigna radiata L.

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Vera Lúcia Arroxelas Galvão de Lima

2004-03-01

Full Text Available OBJETIVO: Considerando a importância dos compostos fenólicos em alimentos e que o broto de feijão-mungo vem sendo incluído na culinária brasileira, este estudo teve como objetivo quantificar o teor de fenólicos totais deste vegetal e avaliar a ação antioxidante do seu extrato aquoso. MÉTODOS: Os compostos fenólicos foram extraídos por quatro sistemas de solventes e dois métodos de extração, os quais foram diferenciados no tempo (2 e 1h e número de extrações (2 e 3 extrações. Os fenólicos totais dos extratos foram quantificados por método espectrofotométrico. RESULTADOS: Os extratos obtidos com água à temperatura ambiente (28ºC, nos dois métodos de extração, foram os que apresentaram maior quantidade de fenólicos totais, sem contudo apresentar diferença significativa entre eles. O método II, que consistiu de três extrações em 1h, pode ser considerado o melhor por ter utilizado menor tempo de extração. O extrato aquoso em sistema modelo b-caroteno/ácido linoléico exibiu ação antioxidante (48,07% de inibição da oxidação, entretanto foi inferior ao padrão BHT. CONCLUSÃO: O broto de feijão-mungo possui considerável quantidade de fenólicos totais, compostos responsáveis por sua ação antioxidante, cujo consumo pode proporcionar efeitos benéficos à saúde.OBJECTIVE: Considering the importance of phenolic compounds in foods and the increasing consumption of mung bean sprouts in Brazil, this study had the objective of quantifying the total phenolic content in this vegetable and to assess the antioxidant activity of its aqueous extract. METHODS: The phenolic compounds were extracted by four solvent systems and two extraction methods, which were different in time (2 and 1h and in number of extractions (2 and 3 extractions. The total phenolic content of the extracts were quantified by the spectrophotometric method. RESULTS: The extracts obtained with water at room temperature (28ºC in both extraction
Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees.

Science.gov (United States)

Peer, Reut; Rivlin, Gil; Golobovitch, Sara; Lapidot, Moshe; Gal-On, Amit; Vainstein, Alexander; Tzfira, Tzvi; Flaishman, Moshe A

2015-04-01

Targeting a gene in apple or fig with ZFN, introduced by transient or stable transformation, should allow genome editing with high precision to advance basic science and breeding programs. Genome editing is a powerful tool for precise gene manipulation in any organism; it has recently been shown to be of great value for annual plants. Classical breeding strategies using conventional cross-breeding and induced mutations have played an important role in the development of new cultivars in fruit trees. However, fruit-tree breeding is a lengthy process with many limitations. Efficient and widely applied methods for targeted modification of fruit-tree genomes are not yet available. In this study, transgenic apple and fig lines carrying a zinc-finger nuclease (ZFNs) under the control of a heat-shock promoter were developed. Editing of a mutated uidA gene, following expression of the ZFN genes by heat shock, was confirmed by GUS staining and PCR product sequencing. Finally, whole plants with a repaired uidA gene due to deletion of a stop codon were regenerated. The ZFN-mediated gene modifications were stable and passed onto regenerants from ZFN-treated tissue cultures. This is the first demonstration of efficient and precise genome editing, using ZFN at a specific genomic locus, in two different perennial fruit trees-apple and fig. We conclude that targeting a gene in apple or fig with a ZFN introduced by transient or stable transformation should allow knockout of a gene of interest. Using this technology for genome editing allows for marker gene-independent and antibiotic selection-free genome engineering with high precision in fruit trees to advance basic science as well as nontransgenic breeding programs.
Método para a Determinação de Ácidos Fenólicos na Parede Celular de Forragens Method for Phenolic Acid Determination in Forage Cell Wall

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Francisco Carlos Deschamps

2002-07-01

Full Text Available Há fatores que limitam a digestão das forragens tropicais e estão associados à dinâmica dos ácidos fenólicos da parede celular. Os estudos destes compostos em forragens podem ser facilitados pela disponibilidade de métodos sensíveis que permitam o processamento de grande número de amostras. No presente trabalho, descreve-se um método para a determinação de ácidos fenólicos na parede celular de forragens, utilizando cromatografia líquida de alta eficiência (CLAE. Bagaço de cana, capim-elefante e folhas de mandioca foram utilizados como amostras experimentais. Para remover substâncias solúveis de baixa massa molecular, foram testados etanol 80% e o detergente neutro, determinando seus efeitos sobre a recuperação das moléculas e benefícios no perfil cromatográfico. Para a obtenção dos ácidos fenólicos livres, as amostras foram solubilizadas em NaOH 1 mol/L, 20ºC por 24 horas. O método proposto foi adequado para a determinação de ácidos fenólicos, apresentando grande sensibilidade e produtividade no laboratório. Para minimizar os efeitos negativos da formação de sal resultante da neutralização ácida do extrato alcalino, sugere-se a diluição da amostra ou a injeção de pequeno volume (5 uL no aparelho. O efeito da utilização de solventes como etanol 80% ou detergente neutro é distinto sobre as amostras das gramíneas e leguminosas. A quantidade de extrativos nas folhas de mandioca foi superior a do bagaço de cana e capim-elefante. A concentração de ácidos fenólicos foi pouco alterada pela ação dos solventes, sendo maior nas amostras de bagaço de cana e capim-elefante, em relação às folhas de mandioca. O método apresentado constitui-se em uma importante ferramenta para o estudo dos ácidos fenólicos na parede celular de forragens tropicais.Factors that limit the digestion of tropical forages are associated to the dynamic of cell wall phenolic acids. The study of these compounds in forages may
Estudio nacional exploratorio descriptivo sobre el fenómeno de trata de personas en Colombia.

OpenAIRE

2009-01-01

Para el Ministerio del Interior y de Justicia como Secretaría Técnica del Comité Interinstitucional para la lucha contra la Trata de Personas y para la Oficina de Naciones Unidas contra la Droga y el Delito en Colombia UNODC, es un honor presentar a ustedes en esta oportunidad el primer Estudio Nacional Exploratorio Descriptivo sobre el Fenómeno de la Trata de Personas en Colombia, desarrollado por unequipo de Investigadoras e Investigadores de la Escuela de Estudios de Género de la Universid...
Polpa de juçara: fonte de compostos fenólicos, aumento da atividade antioxidante e da viabilidade de bactérias probióticas de iogurte

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Sabrina Torres Leite

Full Text Available RESUMO A adição, em iogurtes, de polpa de Juçara como fonte de compostos fenólicos pode modificar as características físico-químicas, contribuir para atividade antioxidante, e interferir na viabilidade de bactérias probióticas e na aceitação sensorial. Com o objetivo de verificar o efeito da adição de polpa de Juçara em iogurte, foi realizado um experimento, em delineamento inteiramente casualizado, em parcelas subdivididas. A parcela principal foi o teor de polpa, com seis níveis (0, 5, 10, 15, 20 e 25% e, como subparcela, o tempo de armazenamento, a 5 oC, com cinco níveis (1, 7, 14, 21 e 28 dias. Os teores percentuais médios de gordura, cinzas, extrato seco total e acidez dos iogurtes não diferiram (P ≥ 0,05. O teor de proteína diminuiu com o aumento do teor de polpa, variando de 4,07% (F0 a 2,9% (F25%. O conteúdo fenólico total e a atividade antioxidante foram maiores no iogurte com 25% de polpa, atingindo valores de 117,84 mg de AGE/100g e 6,95 µmol Trolox/g, respectivamente, assim como a contagem de bactérias probióticas, que variou de 3,17 log UFC.g-1 (F0 a 6,34 log UFC.g-1 (F25%, nos iogurtes, após 28 dias de armazenamento. A adição da polpa de Juçara favoreceu a multiplicação e a viabilidade de bactérias probióticas, contribuiu para o aumento do conteúdo fenólico total e para maior atividade antioxidante dos iogurtes, que apresentaram boa aceitação sensorial pelo consumidor.
Characterization of glucagon-like peptide-1 receptor-binding determinants.

Science.gov (United States)

Xiao, Q; Jeng, W; Wheeler, M B

2000-12-01

Glucagon-like peptide 1 (GLP-1) is a potent insulinotropic hormone currently under study as a therapeutic agent for type 2 diabetes. Since an understanding of the molecular mechanisms leading to high-affinity receptor (R) binding and activation may facilitate the development of more potent GLP-1R agonists, we have localized specific regions of GLP-1R required for binding. The purified N-terminal fragment (hereafter referred to as NT) of the GLP-1R produced in either insect (Sf9) or mammalian (COS-7) cells was shown to bind GLP-1. The physical interaction of NT with GLP-1 was first demonstrated by cross-linking ((125)I-GLP-1/NT complex band at approximately 28 kDa) and secondly by attachment to Ni(2+)-NTA beads. The GLP-1R NT protein attached to beads bound GLP-1, but with lower affinity (inhibitory concentration (IC(50)): 4.5 x 10(-7) M) than wild-type (WT) GLP-1R (IC(50): 5.2 x 10(-9)M). The low affinity of GLP-1R NT suggested that other receptor domains may contribute to GLP-1 binding. This was supported by studies using chimeric glucose-dependent insulinotropic polypeptide (GIP)/GLP-1 receptors. GIP(1-151)/GLP-1R, but not GIP(1-222)/GLP-1R, exhibited specific GLP-1 binding and GLP-1-induced cAMP production, suggesting that the region encompassing transmembrane (TM) domain 1 through to TM3 was required for binding. Since it was hypothesized that certain charged or polar amino acids in this region might be involved in binding, these residues (TM2-TM3) were analyzed by substitution mutagenesis. Five mutants (K197A, D198A, K202A, D215A, R227A) displayed remarkably reduced binding affinity. These studies indicate that the NT domain of the GLP-1R is able to bind GLP-1, but charged residues concentrated at the distal TM2/extracellular loop-1 (EC1) interface (K197, D198, K202) and in EC1 (D215 and R227) probably contribute to the binding determinants of the GLP-1R.
Efeitos da concentração dos compostos fenólicos no mosto da fermentação alcoólica

OpenAIRE

Henrique, Míriam Roberta [UNESP

2013-01-01

O objetivo deste trabalho foi avaliar a relação da concentração dos compostos fenólicos do mosto com a viabilidade celular, viabilidade de brotamento, brotamento celular da levedura alcoólica e a eficiência da fermentação por subprodutos durante a safra 2011/2012 na Usina São Manoel, São Manuel (SP). Esta unidade iniciou a safra com a levedura selecionada CAT-1. Durante a safra, as cepas nativas adentraram o processo fermentativo e também foram avaliadas quanto à sensibilidade a concentração ...
Factors Influencing Mesostigmata Mites (Acari, Parasitiformes In The Alkaline Fen Habitats

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Salmane Ineta

2015-04-01

Full Text Available Mesostigmata mite fauna was investigated in soil of one alkaline fen at Apšuciems in the Maritime Lowland in Latvia in 2009. Thirty sample plots were selected in the following way: half of them were located in Brown Bog Rush, Schoenus ferrugineus and half — in Saw-Sedge, Cladium mariscus prevailing habitat. Soil samples were collected using a soil borer. Vegetation cover was described in accordance with Braun-Blanquet classes. Mites were extracted using modified Berlese funnels. In total, more than 28 species were recorded, of them Prozercon kochi and Pergamasus vagabundus were dominant species. Mean density of Mesostigmata mites ranged from 520 to 2140 ind./m2. Mite abundance and distribution between habitats depended on vegetation cover of the vascular plants, while moss cover and soil pH had no significant influence.
Bloqueio do receptor de leucotrieno CysLT1 em pacientes com fenômeno de Raynaud: evidência capilaroscópica do papel do leucotrieno CysLT1 receptor inhibition in patients with Raynaud's phenomenon: capillaroscopic evidence of the role of leukotriene

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Mario Newton Leitão de Azevedo

2012-02-01

Full Text Available OBJETIVO: Avaliar a ação do medicamento inibidor do receptor de leucotrieno CysLT1 (montelucaste nas alterações vasculares das mãos em pacientes com fenômeno de Raynaud. MÉTODOS: Foram selecionadas pacientes com fenômeno de Raynaud secundário à doença inflamatória do tecido conjuntivo, excluindo tabagismo, hipertensão arterial e diabetes mellitus. As pacientes mantiveram a medicação prévia e iniciaram o uso de montelucaste 10 mg/dia por 60 dias. Foi realizada capilaroscopia periungueal dos dedos das mãos antes do uso da medicação e após 30 e 60 dias. A análise estatística foi feita por meio de porcentagem, média, desvio-padrão e teste exato de Fisher, com intervalo de confiança de 95%. RESULTADOS: Foram estudadas cinco pacientes mulheres, brancas, com fenômeno de Raynaud secundário a doenças do tecido conjuntivo, das quais três apresentavam esclerodermia e duas apresentavam doença mista do tecido conjuntivo. A média de idade foi de 42,4 ± 12,4 anos, e a média de tempo de doença foi de 9,6 ± 4,8 anos. As pacientes estavam em uso de até 20 mg/dia de prednisona (pacientes com doença mista do tecido conjuntivo, nifedipina, pentoxifilina. As medicações foram mantidas. Após o uso de inibidor de receptor de leucotrieno por dois meses, o controle com capilaroscopia ungueal demonstrou diminuição do edema e da palidez e normalização do número, tamanho e distribuição dos capilares. CONCLUSÃO: O uso do montelucaste modificou as alterações capilares observadas na capilaroscopia periungueal de pacientes com fenômeno de RaynaudOBJECTIVE: To assess the effect of the leukotriene receptor inhibitor (montelukast on vascular alterations in fingers of patients with Raynaud's phenomenon. METHODS: Patients with Raynaud's phenomenon of the hands secondary to inflammatory connective tissue disease were selected, and those with the following characteristics were excluded: smokers, arterial hypertension, and diabetes
Full GHG balance of drained fen peatland cropped to spring barley and reed canary grass using comparative assessment of CO2 fluxes

DEFF Research Database (Denmark)

Karki, Sandhya; Elsgaard, Lars; Kandel, Tanka Prasad

2015-01-01

Empirical greenhouse gas (GHG) flux estimates from diverse peatlands are required in order to derive emission factors for managed peatlands. This study on a drained fen peatland quantified the annual GHG balance (Carbon dioxide (CO2), nitrous oxide (N2O), methane (CH4), and C exported in crop yield......) from spring barley (SB) and reed canary grass (RCG) using static opaque chambers for GHG flux measurements and biomass yield for indirectly estimating gross primary production (GPP). Estimates of ecosystem respiration (ER) and GPP were compared with more advanced but costly and labor-intensive dynamic...... by static chamber and dynamic chamber methods was similar, particularly when using nonlinear regression techniques for flux calculations. A comparison of GPP derived from aboveground biomass and from measuring net ecosystem exchange (NEE) showed that GPP estimation from biomass might be useful, or serve...
Functional characterization of a conserved archaeal viral operon revealing single-stranded DNA binding, annealing and nuclease activities

DEFF Research Database (Denmark)

Guo, Yang; Kragelund, Birthe Brandt; White, Malcolm F.

2015-01-01

encoding proteins of unknown function and forming an operon with ORF207 (gp19). SIRV2 gp17 was found to be a single-stranded DNA (ssDNA) binding protein different in structure from all previously characterized ssDNA binding proteins. Mutagenesis of a few conserved basic residues suggested a U......-shaped binding path for ssDNA. The recombinant gp18 showed an ssDNA annealing activity often associated with helicases and recombinases. To gain insight into the biological role of the entire operon, we characterized SIRV2 gp19 and showed it to possess a 5'→3' ssDNA exonuclease activity, in addition...... for rudiviruses and the close interaction among the ssDNA binding, annealing and nuclease proteins strongly point to a role of the gene operon in genome maturation and/or DNA recombination that may function in viral DNA replication/repair....
The thioredoxin TRX-1 modulates the function of the insulin-like neuropeptide DAF-28 during dauer formation in Caenorhabditis elegans.

Science.gov (United States)

Fierro-González, Juan Carlos; Cornils, Astrid; Alcedo, Joy; Miranda-Vizuete, Antonio; Swoboda, Peter

2011-01-27

Thioredoxins comprise a conserved family of redox regulators involved in many biological processes, including stress resistance and aging. We report that the C. elegans thioredoxin TRX-1 acts in ASJ head sensory neurons as a novel modulator of the insulin-like neuropeptide DAF-28 during dauer formation. We show that increased formation of stress-resistant, long-lived dauer larvae in mutants for the gene encoding the insulin-like neuropeptide DAF-28 requires TRX-1 acting in ASJ neurons, upstream of the insulin-like receptor DAF-2. Genetic rescue experiments demonstrate that redox-independent functions of TRX-1 specifically in ASJ neurons are needed for the dauer formation constitutive (Daf-c) phenotype of daf-28 mutants. GFP reporters of trx-1 and daf-28 show opposing expression patterns in dauers (i.e. trx-1 is up-regulated and daf-28 is down-regulated), an effect that is not observed in growing L2/L3 larvae. In addition, functional TRX-1 is required for the down-regulation of a GFP reporter of daf-28 during dauer formation, a process that is likely subject to DAF-28-mediated feedback regulation. Our findings demonstrate that TRX-1 modulates DAF-28 signaling by contributing to the down-regulation of daf-28 expression during dauer formation. We propose that TRX-1 acts as a fluctuating neuronal signaling modulator within ASJ neurons to monitor the adjustment of neuropeptide expression, including insulin-like proteins, during dauer formation in response to adverse environmental conditions.
Engineered Viruses as Genome Editing Devices

Science.gov (United States)

Chen, Xiaoyu; Gonçalves, Manuel A F V

2016-01-01

Genome editing based on sequence-specific designer nucleases, also known as programmable nucleases, seeks to modify in a targeted and precise manner the genetic information content of living cells. Delivering into cells designer nucleases alone or together with donor DNA templates, which serve as surrogate homologous recombination (HR) substrates, can result in gene knockouts or gene knock-ins, respectively. As engineered replication-defective viruses, viral vectors are having an increasingly important role as delivery vehicles for donor DNA templates and designer nucleases, namely, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 (CRISPR−Cas9) nucleases, also known as RNA-guided nucleases (RGNs). We review this dual role played by engineered viral particles on genome editing while focusing on their main scaffolds, consisting of lentiviruses, adeno-associated viruses, and adenoviruses. In addition, the coverage of the growing body of research on the repurposing of viral vectors as delivery systems for genome editing tools is complemented with information regarding their main characteristics, pros, and cons. Finally, this information is framed by a concise description of the chief principles, tools, and applications of the genome editing field as a whole. PMID:26336974
8 CFR 312.1 - Literacy requirements.

Science.gov (United States)

2010-01-01

... 8 Aliens and Nationality 1 2010-01-01 2010-01-01 false Literacy requirements. 312.1 Section 312.1... FOR NATURALIZATION § 312.1 Literacy requirements. (a) General. Except as otherwise provided in... determining English proficiency, as outlined in paragraph(c) of this section. (c) Literacy examination—(1...
Mapeamento do fenômeno de pulsações não-radiais no DHR

Science.gov (United States)

Waelkens, A. H.; Janot Pacheco, E.

2003-08-01

Neste trabalho de IC, pretende-se estabelecer um mapa das características do fenômeno das pulsações não-radiais (PNR) no diagrama HR (DHR). Trata-se de oscilações que não mantém a homotecia radial. O fenômeno foi inicialmente descoberto no Sol nos anos 60 e hoje é detectado num grande número de objetos. Sua grande importância reside em que as pulsações descrevem a física da cavidade em que se propagam. As PNR são classificadas basicamente por suas frequências, amplitudes e certos números quânticos associados. Com o objetivo de descrever a variação desses parâmetros no DHR, procedeu-se a um extenso levantamento bibliográfico (artigos de revistas e outras fontes) cobrindo o tema. Compôs-se assim uma tabela, que será apresentada nesta comunicação, contendo os parâmetros físicos das estrelas (T, L, logg, M) e suas características de PNR, deduzidas a partir de observações fotométricas e/ou espectroscópicas. No momento, completamos os dados sobre as Anãs Brancas e Beta Cep. Apresentaremos no trabalho diagramas L-T com os últimos resultados obtidos, eventualmente com uma terceira dimensão que descreva características pulsacionais.
Association between Insulin Like Growth Factor-1 (IGF-1) gene ...

African Journals Online (AJOL)

The insulin-like growth factor-1 (IGF1) is a key regulator of muscle development and metabolism in birds and other vertebrate. Our objective was to determine the association between IGF1 gene polymorphism and carcass traits in FUNAAB Alpha chicken. Genomic DNA was extracted from the blood of 50 normal feathered ...
Requirement of Xmsx-1 in the BMP-triggered ventralization of Xenopus embryos.

Science.gov (United States)

Yamamoto, T S; Takagi, C; Ueno, N

2000-03-01

Signaling triggered by polypeptide growth factors leads to the activation of their target genes. Several homeobox genes are known to be induced in response to polypeptide growth factors in early Xenopus development. In particular, Xmsx-1, an amphibian homologue of vertebrate Msx-1, is well characterized as a target gene of bone morphogenetic protein (BMP). Here, using a dominant-negative form of Xmsx-1 (VP-Xmsx-1), which is a fusion protein made with the virus-derived VP16 activation domain, we have examined whether Xmsx-1 activity is required in the endogenous ventralizing pathway. VP-Xmsx-1 induced a secondary body axis, complete with muscle and neural tissues, when overexpressed in ventral blastomeres, suggesting that Xmsx-1 activity is necessary for both mesoderm and ectoderm to be ventralized. We have also examined the epistatic relationship between Xmsx-1 and another ventralizing homeobox protein, Xvent-1, and show that Xmsx-1 is likely to be acting upstream of Xvent-1. We propose that Xmsx-1 is required in the BMP-stimulated ventralization pathway that involves the downstream activation of Xvent-1.
Ecoenzymatic stoichiometry and microbial processing of organic matter in northern bogs and fens reveals a common P limitation among peatland types

Science.gov (United States)

We compared C, N, and P concentrations in atmospheric deposition, runoff, and soil standing stocks with microbial respiration (DHA) and ecoenzyme activity (EEA) in an ombrotrophic bog (S2) and a minerotrophic fen (S3) to investigate the environmental drivers of biogeochemical cyc...

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