The modification of siRNA with 3' cholesterol to increase nuclease protection and suppression of native mRNA by select siRNA polyplexes.

PubMed

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.

Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases.

PubMed

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.

Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases

PubMed Central

Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

2015-01-01

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. PMID:26501275

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

PubMed

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.

The Modification of siRNA with 3′ Cholesterol to Increase Nuclease Protection and Suppression of Native mRNA by Select siRNA Polyplexes

PubMed Central

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

A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fichorova, Raina N., E-mail: rfichorova@rics.bwh.harvard.edu; Mendonca, Kevin; Yamamoto, Hidemi S.

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 vascularmore » 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. �

The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis

PubMed Central

Ipsaro, Jonathan J.; Haase, Astrid D.; Knott, Simon R.; Joshua-Tor, Leemor; Hannon, Gregory J.

2012-01-01

PIWI-family proteins and their associated small RNAs (piRNAs) act in an evolutionarily conserved innate immune mechanism that provides an essential protection for germ cell genomes against the activity of mobile genetic elements1. piRNA populations comprise a molecular definition of transposons that permits them to be distinguished from host genes and selectively silenced. piRNAs can be generated in two distinct ways. Primary piRNAs emanate from discrete genomic loci, termed piRNA clusters, and appear to be derived from long, single-stranded precursors2. The biogenesis of primary piRNAs involves at least two nucleolytic steps. An unknown enzyme cleaves piRNA cluster transcripts to generate monophosphorylated piRNA 5' ends. piRNA 3' ends are likely formed by exonucleolytic trimming, after a piRNA precursor is loaded into its PIWI partner1,3. Secondary piRNAs arise during the adaptive ping-pong cycle, with their 5' termini being formed by the activity of PIWIs themselves2,4. A number of proteins have been implicated genetically in primary piRNA biogenesis. One of these, Zucchini, is a member of the phospholipase D family of phosphodiesterases, which includes both phospholipases and nucleases5–7. We have produced a dimeric, soluble fragment of the mouse Zucchini homolog (mZuc/PLD6) and have shown that it possesses single strand-specific nuclease activity. A crystal structure of mZuc at 1.75 Å resolution indicates greater architectural similarity to PLD-family nucleases than to phospholipases. Considered together, our data suggest that the Zucchini proteins act in primary piRNA biogenesis as nucleases, perhaps generating the 5' ends of primary piRNAs. PMID:23064227

[Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis].

PubMed

Liao, Chengshui; Wang, Xiaoli; Tian, Wenjing; Zhang, Mengke; Zhang, Chunjie; Li, Yinju; Wu, Tingcai; Cheng, Xiangchao

2017-08-25

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

Non-viral delivery of genome-editing nucleases for gene therapy.

PubMed

Wang, M; Glass, Z A; Xu, Q

2017-03-01

Manipulating the genetic makeup of mammalian cells using programmable nuclease-based genome-editing technology has recently evolved into a powerful avenue that holds great potential for treating genetic disorders. There are four types of genome-editing nucleases, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and clustered, regularly interspaced, short palindromic repeat-associated nucleases such as Cas9. These nucleases have been harnessed to introduce precise and specific changes of the genome sequence at virtually any genome locus of interest. The therapeutic relevance of these genome-editing technologies, however, is challenged by the safe and efficient delivery of nuclease into targeted cells. Herein, we summarize recent advances that have been made on non-viral delivery of genome-editing nucleases. In particular, we focus on non-viral delivery of Cas9/sgRNA ribonucleoproteins for genome editing. In addition, the future direction for developing non-viral delivery of programmable nucleases for genome editing is discussed.

Nuclease footprint analyses of the interactions between RNase P ribozyme and a model mRNA substrate.

PubMed Central

Trang, P; Hsu, A W; Liu, F

1999-01-01

RNase P ribozyme cleaves an RNA helix substrate which resembles the acceptor stem and T-stem structures of its natural tRNA substrate. By linking the ribozyme covalently to a sequence (guide sequence) complementary to a target RNA, the catalytic RNA can be converted into a sequence-specific ribozyme, M1GS RNA. We have previously shown that M1GS RNA can efficiently cleave the mRNA sequence encoding thymidine kinase (TK) of herpes simplex virus 1. In this study, a footprint procedure using different nucleases was carried out to map the regions of a M1GS ribozyme that potentially interact with the TK mRNA substrate. The ribozyme regions that are protected from nuclease degradation in the presence of the TK mRNA substrate include those that interact with the acceptor stem and T-stem, the 3' terminal CCA sequence and the cleavage site of a tRNA substrate. However, some of the protected regions (e.g. P13 and P14) are unique and not among those protected in the presence of a tRNA substrate. Identification of the regions that interact with a mRNA substrate will allow us to study how M1GS RNA recognizes a mRNA substrate and facilitate the development of mRNA-cleaving ribozymes for gene-targeting applications. PMID:10556315

Halophilic Nuclease from a Moderately Halophilic Micrococcus varians

PubMed Central

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon

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 chosenmore » 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.« less

Genome Editing in Rats Using TALE Nucleases.

PubMed

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.

TaqMan 5′-Nuclease Human Immunodeficiency Virus Type 1 PCR Assay with Phage-Packaged Competitive Internal Control for High-Throughput Blood Donor Screening

PubMed Central

Drosten, C.; Seifried, E.; Roth, W. K.

2001-01-01

Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5′-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5′-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the gag genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5′-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a ≥95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1. PMID:11724836

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

PubMed

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gwon, Gwang Hyeon; Kim, Youngran; Liu, Yaqi

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 domainmore » 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.« less

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

PubMed

Mashimo, Tomoji

2014-01-01

The laboratory rat has been widely used as an animal model in biomedical science for more than 150 years. Applying zinc-finger nucleases or transcription activator-like effector nucleases to rat embryos via microinjection is an efficient genome editing tool for generating targeted knockout rats. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonucleases have been used as an effective tool for precise and multiplex genome editing in mice and rats. In this review, the advantages and disadvantages of these site-specific nuclease technologies for genetic analysis and manipulation in rats are discussed. © 2013 The Author Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Targeting of a Nuclease to Murine Leukemia Virus Capsids Inhibits Viral Multiplication

NASA Astrophysics Data System (ADS)

Natsoulis, Georges; Seshaiah, Partha; Federspiel, Mark J.; Rein, Alan; Hughes, Stephen H.; Boeke, Jef D.

1995-01-01

Capsid-targeted viral inactivation is an antiviral strategy in which toxic fusion proteins are targeted to virions, where they inhibit viral multiplication by destroying viral components. These fusion proteins consist of a virion structural protein moiety and an enzymatic moiety such as a nuclease. Such fusion proteins can severely inhibit transposition of yeast retrotransposon Ty1, an element whose transposition mechanistically resembles retroviral multiplication. We demonstrate that expression of a murine retrovirus capsid-staphylococcal nuclease fusion protein inhibits multiplication of the corresponding murine leukemia virus by 30- to 100-fold. Staphylococcal nuclease is apparently inactive intracellularly and hence nontoxic to the host cell, but it is active extracellularly because of its requirement for high concentrations of Ca2+ ions. Virions assembled in and shed from cells expressing the fusion protein contain very small amounts of intact viral RNA, as would be predicted for nuclease-mediated inhibition of viral multiplication.

Cold denaturation and 2H2O stabilization of a staphylococcal nuclease mutant.

PubMed Central

Antonino, L C; Kautz, R A; Nakano, T; Fox, R O; Fink, A L

1991-01-01

Cold denaturation is now recognized as a general property of proteins but has been observed only under destabilizing conditions, such as moderate denaturant concentration or low pH. By destabilizing the protein using site-directed mutagenesis, we have observed cold denaturation at pH 7.0 in the absence of denaturants in a mutant of staphylococcal nuclease, which we call NCA S28G for a hybrid protein between staphylococcal nuclease and concanavalin A in which there is the point mutation Ser-28----Gly. The temperature of maximum stability (tmax) as determined by circular dichroism (CD) was 18.1 degrees C, and the midpoints of the thermal unfolding transitions (tm) were 0.6 degrees C and 30.0 degrees C. These values may be compared with the tm of 52.5 degrees C for wild-type staphylococcal nuclease, for which no cold denaturation was observed under these conditions. When the stability of the mutant was examined in 2H2O by NMR, CD, or fluorescence, a substantial increase in the amount of folded protein at the tmax was noted as well as a decrease in tmax, reflecting increased stability. PMID:1652762

Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN.

PubMed

Ul Ain, Qurrat; Chung, Jee Young; Kim, Yong-Hee

2015-05-10

Gene therapy by engineered nucleases is a genetic intervention being investigated for curing the hereditary disorders by targeting selected genes with specific nucleotides for establishment, suppression, abolishment of a function or correction of mutation. Here, we review the fast developing technology of targeted genome engineering using site specific programmable nucleases zinc finger nucleases (ZFNs), transcription activator like nucleases (TALENs) and cluster regulatory interspaced short palindromic repeat/CRISPR associated proteins (CRISPR/Cas) based RNA-guided DNA endonucleases (RGENs) and their different characteristics including pros and cons of genome modifications by these nucleases. We have further discussed different types of delivery methods to induce gene editing, novel development in genetic engineering other than nucleases and future prospects. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed

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.

Design of a colicin E7 based chimeric zinc-finger nuclease

NASA Astrophysics Data System (ADS)

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.

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

PubMed

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

2014-01-01

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

Label-free optical detection of single-base mismatches by the combination of nuclease and gold nanoparticles.

PubMed

Liu, Meiying; Yuan, Min; Lou, Xinhui; Mao, Hongju; Zheng, Dongmei; Zou, Ruxing; Zou, Nengli; Tang, Xiangrong; Zhao, Jianlong

2011-07-15

We report here an optical approach that enables highly selective and colorimetric single-base mismatch detection without the need of target modification, precise temperature control or stringent washes. The method is based on the finding that nucleoside monophosphates (dNMPs), which are digested elements of DNA, can better stabilize unmodified gold nanoparticles (AuNPs) than single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with the same base-composition and concentration. The method combines the exceptional mismatch discrimination capability of the structure-selective nucleases with the attractive optical property of AuNPs. Taking S1 nuclease as one example, the perfectly matched 16-base synthetic DNA target was distinctively differentiated from those with single-base mutation located at any position of the 16-base synthetic target. Single-base mutations present in targets with varied length up to 80-base, located either in the middle or near to the end of the targets, were all effectively detected. In order to prove that the method can be potentially used for real clinic samples, the single-base mismatch detections with two HBV genomic DNA samples were conducted. To further prove the generality of this method and potentially overcome the limitation on the detectable lengths of the targets of the S1 nuclease-based method, we also demonstrated the use of a duplex-specific nuclease (DSN) for color reversed single-base mismatch detection. The main limitation of the demonstrated methods is that it is limited to detect mutations in purified ssDNA targets. However, the method coupled with various convenient ssDNA generation and purification techniques, has the potential to be used for the future development of detector-free testing kits in single nucleotide polymorphism screenings for disease diagnostics and treatments. Copyright © 2011 Elsevier B.V. All rights reserved.

Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9

PubMed Central

Koo, Taeyoung; Lee, Jungjoon; Kim, Jin-Soo

2015-01-01

Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations. PMID:25985872

WldS but not Nmnat1 protects dopaminergic neurites from MPP+ neurotoxicity.

PubMed

Antenor-Dorsey, Jo Ann V; O'Malley, Karen L

2012-02-08

The WldS mouse mutant ("Wallerian degeneration-slow") delays axonal degeneration in a variety of disorders including in vivo models of Parkinson's disease. The mechanisms underlying WldS -mediated axonal protection are unclear, although many studies have attributed WldS neuroprotection to the NAD+-synthesizing Nmnat1 portion of the fusion protein. Here, we used dissociated dopaminergic cultures to test the hypothesis that catalytically active Nmnat1 protects dopaminergic neurons from toxin-mediated axonal injury. Using mutant mice and lentiviral transduction of dopaminergic neurons, the present findings demonstrate that WldS but not Nmnat1, Nmnat3, or cytoplasmically-targeted Nmnat1 protects dopamine axons from the parkinsonian mimetic N-methyl-4-phenylpyridinium (MPP+). Moreover, NAD+ synthesis is not required since enzymatically-inactive WldS still protects. In addition, NAD+ by itself is axonally protective and together with WldS is additive in the MPP+ model. Our data suggest that NAD+ and WldS act through separate and possibly parallel mechanisms to protect dopamine axons. As MPP+ is thought to impair mitochondrial function, these results suggest that WldS might be involved in preserving mitochondrial health or maintaining cellular metabolism.

Apollo contributes to G overhang maintenance and protects leading-end telomeres.

PubMed

Wu, Peng; van Overbeek, Megan; Rooney, Sean; de Lange, Titia

2010-08-27

Mammalian telomeres contain a single-stranded 3' overhang that is thought to mediate telomere protection. Here we identify the TRF2-interacting factor Apollo as a nuclease that contributes to the generation/maintenance of this overhang. The function of mouse Apollo was determined using Cre-mediated gene deletion, complementation with Apollo mutants, and the TRF2-F120A mutant that cannot bind Apollo. Cells lacking Apollo activated the ATM kinase at their telomeres in S phase and showed leading-end telomere fusions. These telomere dysfunction phenotypes were accompanied by a reduction in the telomeric overhang signal. The telomeric functions of Apollo required its TRF2-interaction and nuclease motifs. Thus, TRF2 recruits the Apollo nuclease to process telomere ends synthesized by leading-strand DNA synthesis, thereby creating a terminal structure that avoids ATM activation and resists end-joining. These data establish that the telomeric overhang is required for the protection of telomeres from the DNA damage response. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Structural characterization of the virulence factor nuclease A from Streptococcus agalactiae

DOE PAGES

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 structuremore » 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. Lastly, 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.« less

Enhanced gene disruption by programmable nucleases delivered by a minicircle vector.

PubMed

Dad, A-B K; Ramakrishna, S; Song, M; Kim, H

2014-11-01

Targeted genetic modification using programmable nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) is of great value in biomedical research, medicine and biotechnology. Minicircle vectors, which lack extraneous bacterial sequences, have several advantages over conventional plasmids for transgene delivery. Here, for the first time, we delivered programmable nucleases into human cells using transient transfection of a minicircle vector and compared the results with those obtained using a conventional plasmid. Surrogate reporter assays and T7 endonuclease analyses revealed that cells in the minicircle vector group displayed significantly higher mutation frequencies at the target sites than those in the conventional plasmid group. Quantitative PCR and reverse transcription-PCR showed higher vector copy number and programmable nuclease transcript levels, respectively, in 293T cells after minicircle versus conventional plasmid vector transfection. In addition, tryphan blue staining and flow cytometry after annexin V and propidium iodide staining showed that cell viability was also significantly higher in the minicircle group than in the conventional plasmid group. Taken together, our results show that gene disruption using minicircle vector-mediated delivery of ZFNs and TALENs is a more efficient, safer and less toxic method than using a conventional plasmid, and indicate that the minicircle vector could serve as an advanced delivery method for programmable nucleases.

SNMIB/Apollo protects leading-strand telomeres against NHEJ-mediated repair.

PubMed

Lam, Yung C; Akhter, Shamima; Gu, Peili; Ye, Jing; Poulet, Anaïs; Giraud-Panis, Marie-Josèphe; Bailey, Susan M; Gilson, Eric; Legerski, Randy J; Chang, Sandy

2010-07-07

Progressive telomere attrition or deficiency of the protective shelterin complex elicits a DNA damage response as a result of a cell's inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. SNMIB/Apollo is a shelterin-associated protein and a member of the SMN1/PSO2 nuclease family that localizes to telomeres through its interaction with TRF2. Here, we generated SNMIB/Apollo knockout mouse embryo fibroblasts (MEFs) to probe the function of SNMIB/Apollo at mammalian telomeres. SNMIB/Apollo null MEFs exhibit an increased incidence of G2 chromatid-type fusions involving telomeres created by leading-strand DNA synthesis, reflective of a failure to protect these telomeres after DNA replication. Mutations within SNMIB/Apollo's conserved nuclease domain failed to suppress this phenotype, suggesting that its nuclease activity is required to protect leading-strand telomeres. SNMIB/Apollo(-/-)ATM(-/-) MEFs display robust telomere fusions when Trf2 is depleted, indicating that ATM is dispensable for repair of uncapped telomeres in this setting. Our data implicate the 5'-3' exonuclease function of SNM1B/Apollo in the generation of 3' single-stranded overhangs at newly replicated leading-strand telomeres to protect them from engaging the non-homologous end-joining pathway.

Genetic correction using engineered nucleases for gene therapy applications.

PubMed

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.

Development of sequence-specific antimicrobials based on programmable CRISPR-Cas nucleases

PubMed Central

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

2014-01-01

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 Cas91, 2 delivered by a bacteriophage. We show that Cas9 re-programmed to target virulence genes kills virulent, but not avirulent, Staphylococcus aureus. Re-programming the nuclease to target antibiotic resistance genes destroys staphylococcal plasmids that harbor antibiotic resistance genes3, 4 and immunizes avirulent staphylococci to prevent the spread of plasmid-borne resistance genes. We also demonstrate the approach in vivo, showing its efficacy against S. aureus in a mouse skin colonization model. This new technology creates opportunities to manipulate complex bacterial populations in a sequence-specific manner. PMID:25282355

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

PubMed

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.

Cell Wall-Anchored Nuclease of Streptococcus sanguinis Contributes to Escape from Neutrophil Extracellular Trap-Mediated Bacteriocidal Activity

PubMed Central

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 Mg2+ and Ca2+ 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. PMID:25084357

DNA aptamers against FokI nuclease domain for genome editing applications.

PubMed

Nishio, Maui; Matsumoto, Daisuke; Kato, Yoshio; Abe, Koichi; Lee, Jinhee; Tsukakoshi, Kaori; Yamagishi, Ayana; Nakamura, Chikashi; Ikebukuro, Kazunori

2017-07-15

Genome editing with site-specific nucleases (SSNs) can modify only the target gene and may be effective for gene therapy. The main limitation of genome editing for clinical use is off-target effects; excess SSNs in the cells and their longevity can contribute to off-target effects. Therefore, a controlled delivery system for SSNs is necessary. FokI nuclease domain (FokI) is a common DNA cleavage domain in zinc finger nuclease (ZFN) and transcription activator-like effector nuclease. Previously, we reported a zinc finger protein delivery system that combined aptamer-fused, double-strand oligonucleotides and nanoneedles. Here, we report the development of DNA aptamers that bind to the target molecules, with high affinity and specificity to the FokI. DNA aptamers were selected in six rounds of systematic evolution of ligands by exponential enrichment. Aptamers F6#8 and #71, which showed high binding affinity to FokI (K d =82nM, 74nM each), showed resistance to nuclease activity itself and did not inhibit nuclease activity. We immobilized the ZFN-fused GFP to nanoneedles through these aptamers and inserted the nanoneedles into HEK293 cells. We observed the release of ZFN-fused GFP from the nanoneedles in the presence of cells. Therefore, these aptamers are useful for genome editing applications such as controlled delivery of SSNs. Copyright © 2016 Elsevier B.V. All rights reserved.

Probing chromatin structure with nuclease sensitivity assays.

PubMed

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.

Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity.

PubMed

Flenker, Katie S; Burghardt, Elliot L; Dutta, Nirmal; Burns, William J; Grover, Julia M; Kenkel, Elizabeth J; Weaver, Tyler M; Mills, James; Kim, Hyeon; Huang, Lingyan; Owczarzy, Richard; Musselman, Catherine A; Behlke, Mark A; Ford, Bradley; McNamara, James O

2017-06-07

Rapid and accurate bacterial detection methods are needed for clinical diagnostic, water, and food testing applications. The wide diversity of bacterial nucleases provides a rich source of enzymes that could be exploited as signal amplifying biomarkers to enable rapid, selective detection of bacterial species. With the exception of the use of micrococcal nuclease activity to detect Staphylococcus aureus, rapid methods that detect bacterial pathogens via their nuclease activities have not been developed. Here, we identify endonuclease I as a robust biomarker for E. coli and develop a rapid ultrasensitive assay that detects its activity. Comparison of nuclease activities of wild-type and nuclease-knockout E. coli clones revealed that endonuclease I is the predominant DNase in E. coli lysates. Endonuclease I is detectable by immunoblot and activity assays in uropathogenic E. coli strains. A rapid assay that detects endonuclease I activity in patient urine with an oligonucleotide probe exhibited substantially higher sensitivity for urinary tract infections than that reported for rapid urinalysis methods. The 3 hr turnaround time is much shorter than that of culture-based methods, thereby providing a means for expedited administration of appropriate antimicrobial therapy. We suggest this approach could address various unmet needs for rapid detection of E. coli. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

An ultrasensitive fluorescence method suitable for quantitative analysis of mung bean nuclease and inhibitor screening in vitro and vivo.

PubMed

Peng, Lan; Fan, Jialong; Tong, Chunyi; Xie, Zhenhua; Zhao, Chuan; Liu, Xuanming; Zhu, Yonghua; Liu, Bin

2016-09-15

Mung bean nuclease is a single stranded specific DNA and RNA endonuclease purified from mung bean sprouts. It yields 5'-phosphate terminated mono- and oligonucleotides. The activity level of this nuclease can act as a marker to monitor the developmental process of mung bean sprouts. In order to facilitate the activity and physiological analysis of this nuclease, we have developed a biosensing assay system based on the mung bean nuclease-induced single-stranded DNA scission and the affinity difference of graphene oxide for single-stranded DNA containing different numbers of bases. This end-point measurement method can detect mung bean nuclease in a range of 2×10(-4) to 4×10(-2) with a detection limit of 1×10(-4) unit/mL. In addition, we demonstrate the utility of the assay for screening chemical antibiotics and metal ions, resulting in the identification of several inhibitors of this enzyme in vitro. Furthermore, we firstly report that inhibiting mung bean nuclease by gentamycin sulfate and kanamycin in vivo can suppress mung bean sprouts growth. In summary, this method provides an alternative tool for the biochemical analysis for mung bean nuclease and indicates the feasibility of high-throughput screening specific inhibitors of this nuclease in vitro and in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Nuclease-mediated genome editing: At the front-line of functional genomics technology.

PubMed

Sakuma, Tetsushi; Woltjen, Knut

2014-01-01

Genome editing with engineered endonucleases is rapidly becoming a staple method in developmental biology studies. Engineered nucleases permit random or designed genomic modification at precise loci through the stimulation of endogenous double-strand break repair. Homology-directed repair following targeted DNA damage is mediated by co-introduction of a custom repair template, allowing the derivation of knock-out and knock-in alleles in animal models previously refractory to classic gene targeting procedures. Currently there are three main types of customizable site-specific nucleases delineated by the source mechanism of DNA binding that guides nuclease activity to a genomic target: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Among these genome engineering tools, characteristics such as the ease of design and construction, mechanism of inducing DNA damage, and DNA sequence specificity all differ, making their application complementary. By understanding the advantages and disadvantages of each method, one may make the best choice for their particular purpose. © 2014 The Authors Development, Growth & Differentiation © 2014 Japanese Society of Developmental Biologists.

Small Molecule Targeted Recruitment of a Nuclease to RNA.

PubMed

Costales, Matthew G; Matsumoto, Yasumasa; Velagapudi, Sai Pradeep; Disney, Matthew D

2018-06-06

The choreography between RNA synthesis and degradation is a key determinant in biology. Engineered systems such as CRISPR have been developed to rid a cell of RNAs. Here, we show that a small molecule can recruit a nuclease to a specific transcript, triggering its destruction. A small molecule that selectively binds the oncogenic microRNA(miR)-96 hairpin precursor was appended with a short 2'-5' poly(A) oligonucleotide. The conjugate locally activated endogenous, latent ribonuclease (RNase L), which selectively cleaved the miR-96 precursor in cancer cells in a catalytic and sub-stoichiometric fashion. Silencing miR-96 derepressed pro-apoptotic FOXO1 transcription factor, triggering apoptosis in breast cancer, but not healthy breast, cells. These results demonstrate that small molecules can be programmed to selectively cleave RNA via nuclease recruitment and has broad implications.

Hybrid nanosensor for colorimetric and ultrasensitive detection of nuclease contaminations

NASA Astrophysics Data System (ADS)

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.

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease.

PubMed

Wolfs, Jason M; Hamilton, Thomas A; Lant, Jeremy T; Laforet, Marcon; Zhang, Jenny; Salemi, Louisa M; Gloor, Gregory B; Schild-Poulter, Caroline; Edgell, David R

2016-12-27

The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.

S1 of distinct IBV population expressed from recombinant adenovirus confers protection against challenge.

PubMed

Toro, H; Zhang, J F; Gallardo, R A; van Santen, V L; van Ginkel, F W; Joiner, K S; Breedlove, C

2014-06-01

Protective properties of three distinct infectious bronchitis virus (IBV) Ark Delmarva poultry industry (ArkDPI) S1 proteins encoded from replication-defective recombinant adenovirus vectors were investigated. Using a suboptimal dose of each recombinant virus, we demonstrated that IBV S1 amino acid sequences showing > or = 95.8% amino acid identity to the S1 of the challenge strain differed in their ability at conferring protection. Indeed, the S1 sequence of the IBV population previously designated C4 (AdIBVS1.C4), which protected the most poorly, differs from the S1 sequence of population C2 (AdIBVS1.C2), which provided the highest protection, only at amino acid position 56. The fact that a change in one amino acid in this region significantly altered the induction of a protective immune response against this protein provides evidence that the first portion of S1 displays relevant immunoprotective epitopes. Use of an optimal dose of AdIBVS1.C2 effectively protected chickens from clinical signs and significantly reduced viral load after IBV Ark virulent challenge. Moreover, increased numbers of both IgA and IgG IBV-specific antibody secreting lymphocytes were detected in the spleen after challenge. The increased response detected for both IgA and IgG lymphocytes after challenge might be explained by vaccine-induced B memory cells. The fact that a single vaccination with Ad/IBVS1.C2 provides protection against IBV challenge is promising, because Ad-vectored vaccines can be mass delivered by in ovo inoculation using automated in ovo injectors.

Design principles for nuclease-deficient CRISPR-based transcriptional regulators

PubMed Central

Jensen, Michael K

2018-01-01

Abstract The engineering of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins continues to expand the toolkit available for genome editing, reprogramming gene regulation, genome visualisation and epigenetic studies of living organisms. In this review, the emerging design principles on the use of nuclease-deficient CRISPR-based reprogramming of gene expression will be presented. The review will focus on the designs implemented in yeast both at the level of CRISPR proteins and guide RNA (gRNA), but will lend due credits to the seminal studies performed in other species where relevant. In addition to design principles, this review also highlights applications benefitting from the use of CRISPR-mediated transcriptional regulation and discusses the future directions to further expand the toolkit for nuclease-deficient reprogramming of genomes. As such, this review should be of general interest for experimentalists to get familiarised with the parameters underlying the power of reprogramming genomic functions by use of nuclease-deficient CRISPR technologies. PMID:29726937

Design principles for nuclease-deficient CRISPR-based transcriptional regulators.

PubMed

Jensen, Michael K

2018-06-01

The engineering of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins continues to expand the toolkit available for genome editing, reprogramming gene regulation, genome visualisation and epigenetic studies of living organisms. In this review, the emerging design principles on the use of nuclease-deficient CRISPR-based reprogramming of gene expression will be presented. The review will focus on the designs implemented in yeast both at the level of CRISPR proteins and guide RNA (gRNA), but will lend due credits to the seminal studies performed in other species where relevant. In addition to design principles, this review also highlights applications benefitting from the use of CRISPR-mediated transcriptional regulation and discusses the future directions to further expand the toolkit for nuclease-deficient reprogramming of genomes. As such, this review should be of general interest for experimentalists to get familiarised with the parameters underlying the power of reprogramming genomic functions by use of nuclease-deficient CRISPR technologies.

Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps.

PubMed

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.

Knockdown of nuclease activity in the gut enhances RNAi efficiency in the Colorado potato beetle, Leptinotarsa decemlineata, but not in the desert locust, Schistocerca gregaria.

PubMed

Spit, Jornt; Philips, Annelies; Wynant, Niels; Santos, Dulce; Plaetinck, Geert; Vanden Broeck, Jozef

2017-02-01

The responsiveness towards orally delivered dsRNA and the potency of a subsequent environmental RNA interference (RNAi) response strongly differs between different insect species. While some species are very sensitive to dsRNA delivery through the diet, others are not. The underlying reasons for this may vary, but degradation of dsRNA by nucleases in the gut lumen is believed to play a crucial role. The Colorado potato beetle, Leptinotarsa decemlineata, is a voracious defoliator of potato crops worldwide, and is currently under investigation for novel control methods based on dsRNA treatments. Here we describe the identification and characterization of two nuclease genes exclusively expressed in the gut of this pest species. Removal of nuclease activity in adults increased the sensitivity towards dsRNA and resulted in improved protection of potato plants. A similar strategy in the desert locust, Schistocerca gregaria, for which we show a far more potent nuclease activity in the gut juice, did however not lead to an improvement of the RNAi response. Possible reasons for this are discussed. Taken together, the present data confirm a negative effect of nucleases in the gut on the environmental RNAi response, and further suggest that interfering with this activity is a strategy worth pursuing for improving RNAi efficacy in insect pest control applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Repair of DNA-polypeptide crosslinks by human excision nuclease

NASA Astrophysics Data System (ADS)

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

Determinants of the cytotoxicity of PrrC anticodon nuclease and its amelioration by tRNA repair

PubMed Central

Meineke, Birthe; Shuman, Stewart

2012-01-01

Breakage of tRNALys(UUU) by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies a host antiviral response to phage T4 infection that is ultimately thwarted by a virus-encoded RNA repair system. PrrC homologs are prevalent in other bacteria, but their activities and substrates are not defined. We find that induced expression of EcoPrrC is toxic in Saccharomyces cerevisiae and E. coli, whereas the Neisseria meningitidis PrrC (NmePrrC) is not. PrrCs consist of an N-terminal NTPase module and a C-terminal nuclease module. Domain swaps identified the EcoPrrC nuclease domain as decisive for toxicity when linked to either the Eco or Nme NTPase. Indeed, a single arginine-to-tryptophan change in the NmePrrC nuclease domain (R316W) educed a gain-of-function and rendered NmePrrC toxic to yeast, with genetic evidence for tRNALys(UUU) being the relevant target. The reciprocal Trp-to-Arg change in EcoPrrC (W335R) abolished its toxicity. Further mutagenesis of the EcoPrrC nuclease domain highlighted an ensemble of 15 essential residues and distinguished between hypomorphic alleles and potential nuclease-nulls. We report that the RNA repair phase of the bacterial virus-host dynamic is also portable to yeast, where coexpression of the T4 enzymes Pnkp and Rnl1 ameliorated the toxicity of NmePrrC-R316W. Plant tRNA ligase AtRNL also countered NmePrrC-R316W toxicity, in a manner that depended on AtRNL's 5′-kinase and ligase functions. PMID:22101242

Determinants of the cytotoxicity of PrrC anticodon nuclease and its amelioration by tRNA repair.

PubMed

Meineke, Birthe; Shuman, Stewart

2012-01-01

Breakage of tRNA(Lys(UUU)) by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies a host antiviral response to phage T4 infection that is ultimately thwarted by a virus-encoded RNA repair system. PrrC homologs are prevalent in other bacteria, but their activities and substrates are not defined. We find that induced expression of EcoPrrC is toxic in Saccharomyces cerevisiae and E. coli, whereas the Neisseria meningitidis PrrC (NmePrrC) is not. PrrCs consist of an N-terminal NTPase module and a C-terminal nuclease module. Domain swaps identified the EcoPrrC nuclease domain as decisive for toxicity when linked to either the Eco or Nme NTPase. Indeed, a single arginine-to-tryptophan change in the NmePrrC nuclease domain (R316W) educed a gain-of-function and rendered NmePrrC toxic to yeast, with genetic evidence for tRNA(Lys(UUU)) being the relevant target. The reciprocal Trp-to-Arg change in EcoPrrC (W335R) abolished its toxicity. Further mutagenesis of the EcoPrrC nuclease domain highlighted an ensemble of 15 essential residues and distinguished between hypomorphic alleles and potential nuclease-nulls. We report that the RNA repair phase of the bacterial virus-host dynamic is also portable to yeast, where coexpression of the T4 enzymes Pnkp and Rnl1 ameliorated the toxicity of NmePrrC-R316W. Plant tRNA ligase AtRNL also countered NmePrrC-R316W toxicity, in a manner that depended on AtRNL's 5'-kinase and ligase functions.

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

PubMed

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

Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2

PubMed Central

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

Geminivirus-Mediated Genome Editing in Potato (Solanum tuberosum L.) Using Sequence-Specific Nucleases

PubMed Central

Butler, Nathaniel M.; Baltes, Nicholas J.; Voytas, Daniel F.; Douches, David S.

2016-01-01

Genome editing using sequence-specific nucleases (SSNs) is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas) for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via non-homologous end-joining (NHEJ) has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination (HR) remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR) was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and a novel approach to gene targeting in a vegetatively propagated species. PMID:27493650

alpha-DNA II. Synthesis of unnatural alpha-anomeric oligodeoxyribonucleotides containing the four usual bases and study of their substrate activities for nucleases.

PubMed Central

Morvan, F; Rayner, B; Imbach, J L; Thenet, S; Bertrand, J R; Paoletti, J; Malvy, C; Paoletti, C

1987-01-01

This paper describes for the first time the synthesis of alpha-oligonucleotides containing the four usual bases. Two unnatural hexadeoxyribonucleotides: alpha-[d(CpApTpGpCpG)] and alpha-[d(CpGpCpApTpG)], consisting only of alpha-anomeric nucleotide units, were obtained by an improved phosphotriester method, in solution. Starting material was the four base-protected alpha-deoxyribonucleosides 3a-d. Pyrimidine alpha-deoxynucleosides 3a and 3b were prepared by self-anomerization reactions followed by selective deprotection of sugar hydroxyles, while the two purine alpha-deoxynucleosides 3c and 3d were prepared by glycosylation reactions. In the case of guanine alpha-nucleoside derivative a supplementary base-protecting group: N,N-diphenylcarbamoyl was introduced on O6-position in order to avoid side-reactions during oligonucleotide assembling. The hexadeoxynucleotide alpha-[d(CpApTpGpCpG)] was tested as substrate of selected endo- and exonucleases. In conditions where the natural corresponding beta-hexamer was completely degradated by nuclease S1 and calf spleen phosphodiesterase, the alpha-oligonucleotide remained almost intact. PMID:3575096

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

PubMed

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.

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

PubMed

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.

Delivery methods for site-specific nucleases: Achieving the full potential of therapeutic gene editing.

PubMed

Liu, Jia; Shui, Sai-Lan

2016-12-28

The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications. However, safe and efficient delivery methods remain the major obstacle for therapeutic gene editing. In this review, we summarize the recent progress on nuclease delivery methods, highlight their impact on the outcomes of gene editing and discuss the potential of different delivery approaches for therapeutic gene editing. Copyright © 2016 Elsevier B.V. All rights reserved.

Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease.

PubMed

Zhang, Guo-Chang; Kong, In Iok; Kim, Heejin; Liu, Jing-Jing; Cate, Jamie H D; Jin, Yong-Su

2014-12-01

Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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

PubMed

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.

Engineering nucleases for gene targeting: safety and regulatory considerations.

PubMed

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.

PubMed

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.

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

PubMed Central

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

2014-01-01

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. PMID:24989021

Cancer translocations in human cells induced by zinc finger and TALE nucleases

PubMed Central

Piganeau, Marion; Ghezraoui, Hind; De Cian, Anne; Guittat, Lionel; Tomishima, Mark; Perrouault, Loic; René, Oliver; Katibah, George E.; Zhang, Lei; Holmes, Michael C.; Doyon, Yannick; Concordet, Jean-Paul; Giovannangeli, Carine; Jasin, Maria; Brunet, Erika

2013-01-01

Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it challenging to assign a specific phenotypic change to a specific aberration. In this study, we set out to use genome editing with zinc finger (ZFN) and transcription activator-like effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and anaplastic large cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1–FLI1 and NPM1–ALK fusions. Breakpoint junctions recovered after ZFN cleavage in human embryonic stem (ES) cell–derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases. PMID:23568838

Identification of Staphylococcal Nuclease Domain-containing 1 (SND1) as a Metadherin-interacting Protein with Metastasis-promoting Functions*

PubMed Central

Blanco, Mario Andres; Alečković, Maša; Hua, Yuling; Li, Tuo; Wei, Yong; Xu, Zhen; Cristea, Ileana M.; Kang, Yibin

2011-01-01

Metastasis is the deadliest and most poorly understood feature of malignant diseases. Recent work has shown that Metadherin (MTDH) is overexpressed in over 40% of breast cancer patients and promotes metastasis and chemoresistance in experimental models of breast cancer progression. Here we applied mass spectrometry-based screen to identify staphylococcal nuclease domain-containing 1 (SND1) as a candidate MTDH-interacting protein. After confirming the interaction between SND1 and MTDH, we tested the role of SND1 in breast cancer and found that it strongly promotes lung metastasis. SND1 was further shown to promote resistance to apoptosis and to regulate the expression of genes associated with metastasis and chemoresistance. Analyses of breast cancer clinical microarray data indicated that high expression of SND1 in primary tumors is strongly associated with reduced metastasis-free survival in multiple large scale data sets. Thus, we have uncovered SND1 as a novel MTDH-interacting protein and shown that it is a functionally and clinically significant mediator of metastasis. PMID:21478147

A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation.

PubMed

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

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. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed

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.

Identification of Plasmodium falciparum DNA Repair Protein Mre11 with an Evolutionarily Conserved Nuclease Function

PubMed Central

Badugu, Sugith Babu; Nabi, Shaik Abdul; Vaidyam, Pratap; Laskar, Shyamasree; Bhattacharyya, Sunanda; Bhattacharyya, Mrinal Kanti

2015-01-01

The eukaryotic Meiotic Recombination protein 11 (Mre11) plays pivotal roles in the DNA damage response (DDR). Specifically, Mre11 senses and signals DNA double strand breaks (DSB) and facilitates their repair through effector proteins belonging to either homologous recombination (HR) or non-homologous end joining (NHEJ) repair mechanisms. In the human malaria parasite Plasmodium falciparum, HR and alternative-NHEJ have been identified; however, little is known about the upstream factors involved in the DDR of this organism. In this report, we identify a putative ortholog of Mre11 in P. falciparum (PfalMre11) that shares 22% sequence similarity to human Mre11. Homology modeling reveals striking structural resemblance of the predicted PfalMre11 nuclease domain to the nuclease domain of Saccharomyces cerevisiae Mre11 (ScMre11). Complementation analyses reveal functional conservation of PfalMre11 nuclease activity as demonstrated by the ability of the PfalMre11 nuclease domain, in conjunction with the C-terminal domain of ScMre11, to functionally complement an mre11 deficient yeast strain. Functional complementation was virtually abrogated by an amino acid substitution in the PfalMre11 nuclease domain (D398N). PfalMre11 is abundant in the mitotically active trophozoite and schizont stages of P. falciparum and is up-regulated in response to DNA damage, suggesting a role in the DDR. PfalMre11 exhibits physical interaction with PfalRad50. In addition, yeast 2-hybrid studies show that PfalMre11 interacts with ScRad50 and ScXrs2, two important components of the well characterized Mre11-Rad50-Xrs2 complex which is involved in DDR signaling and repair in S. cerevisiae, further supporting a role for PfalMre11 in the DDR. Taken together, these findings provide evidence that PfalMre11 is an evolutionarily conserved component of the DDR in Plasmodium. PMID:25938776

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

PubMed

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.

Inactivation of Hepatitis B Virus Replication in Cultured Cells and In Vivo with Engineered Transcription Activator-Like Effector Nucleases

PubMed Central

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

2013-01-01

Chronic hepatitis B virus (HBV) infection remains an important global health problem. Stability of the episomal covalently closed circular HBV DNA (cccDNA) is largely responsible for the modest curative efficacy of available therapy. Since licensed anti-HBV drugs have a post-transcriptional mechanism of action, disabling cccDNA is potentially of therapeutic benefit. To develop this approach, we engineered mutagenic transcription activator-like effector nucleases (TALENs) that target four HBV-specific sites within the viral genome. TALENs with cognate sequences in the S or C open-reading frames (ORFs) efficiently disrupted sequences at the intended sites and suppressed markers of viral replication. Following triple transfection of cultured HepG2.2.15 cells under mildly hypothermic conditions, the S TALEN caused targeted mutation in ~35% of cccDNA molecules. Markers of viral replication were also inhibited in vivo in a murine hydrodynamic injection model of HBV replication. HBV target sites within S and C ORFs of the injected HBV DNA were mutated without evidence of toxicity. These findings are the first to demonstrate a targeted nuclease-mediated disruption of HBV cccDNA. Efficacy in vivo also indicates that these engineered nucleases have potential for use in treatment of chronic HBV infection. PMID:23883864

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein.

PubMed

Pawluk, April; Shah, Megha; Mejdani, Marios; Calmettes, Charles; Moraes, Trevor F; Davidson, Alan R; Maxwell, Karen L

2017-12-12

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. IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system. Copyright © 2017 Pawluk et al.

Comparison of culture and a novel 5' Taq nuclease assay for direct detection of Campylobacter fetus subsp. venerealis in clinical specimens from cattle.

PubMed

McMillen, Lyle; Fordyce, Geoffry; Doogan, Vivienne J; Lew, Ala E

2006-03-01

A Campylobacter fetus subsp. venerealis-specific 5' Taq nuclease PCR assay using a 3' minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5' Taq nuclease assay detected approximately one single cell compared to 100 and 10 cells in the conventional PCR assay and 2,500 and 25,000 cells from selective culture from inoculated smegma and mucus, respectively. The respective detection limits following the enrichments from smegma and mucus were 5,000 and 50 cells/inoculum for the conventional PCR compared to 500 and 50 cells/inoculum for the 5' Taq nuclease assay. Field sampling confirmed the sensitivity and the specificity of the 5' Taq nuclease assay by detecting an additional 40 bulls that were not detected by culture. Urine-inoculated samples demonstrated comparable detection of C. fetus subsp. venerealis by both culture and the 5' Taq nuclease assay; however, urine was found to be less effective than smegma for bull sampling. Three infected bulls were tested repetitively to compare sampling tools, and the bull rasper proved to be the most suitable, as evidenced by the improved ease of specimen collection and the consistent detection of higher levels of C. fetus subsp. venerealis. The 5' Taq nuclease assay demonstrates a statistically significant association with culture (chi2 = 29.8; P < 0.001) and significant improvements for the detection of C. fetus subsp. venerealis-infected animals from crude clinical extracts following prolonged transport.

Structural specificity of Rn nuclease I as probed on yeast tRNA(Phe) and tRNA(Asp).

PubMed Central

Przykorska, A; el Adlouni, C; Keith, G; Szarkowski, J W; Dirheimer, G

1992-01-01

A single-strand-specific nuclease from rye germ (Rn nuclease I) was characterized as a tool for secondary and tertiary structure investigation of RNAs. To test the procedure, yeast tRNA(Phe) and tRNA(Asp) for which the tertiary structures are known, as well as the 3'-half of tRNA(Asp) were used as substrates. In tRNA(Phe) the nuclease introduced main primary cuts at positions U33 and A35 of the anticodon loop and G18 and G19 of the D loop. No primary cuts were observed within the double stranded stems. In tRNA(Asp) the main cuts occurred at positions U33, G34, U35, C36 of the anticodon loop and G18 and C20:1 positions in the D loop. No cuts were observed in the T loop in intact tRNA(Asp) but strong primary cleavages occurred at positions psi 55, C56, A57 within that loop in the absence of the tertiary interactions between T and D loops (use of 3'-half tRNA(Asp)). These results show that Rn nuclease I is specific for exposed single-stranded regions. Images PMID:1542562

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

PubMed

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.

Dna2 nuclease-helicase structure, mechanism and regulation by Rpa

PubMed Central

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

2015-01-01

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. DOI: http://dx.doi.org/10.7554/eLife.09832.001 PMID:26491943

Selective Enhancement of Nucleases by Polyvalent DNA-Functionalized Gold Nanoparticles

PubMed Central

Prigodich, Andrew E.; Alhasan, Ali H.

2011-01-01

We demonstrate that polyvalent DNA-functionalized gold nanoparticles (DNA-Au NPs) selectively enhance Ribonuclease H (RNase H) activity, while inhibiting most biologically relevant nucleases. This combination of properties is particularly interesting in the context of gene regulation, since high RNase H activity results in rapid mRNA degradation and general nuclease inhibition results in high biological stability. We investigate the mechanism of selective RNase H activation and find that the high DNA density of DNA-Au NPs is responsible for this unusual behavior. This work adds to our understanding of polyvalent DNA-Au NPs as gene regulation agents, and suggests a new model for selectively controlling protein-nanoparticle interactions. PMID:21268581

The extracellular nuclease Dns and its role in natural transformation of Vibrio cholerae.

PubMed

Blokesch, Melanie; Schoolnik, Gary K

2008-11-01

Free extracellular DNA is abundant in many aquatic environments. While much of this DNA will be degraded by nucleases secreted by the surrounding microbial community, some is available as transforming material that can be taken up by naturally competent bacteria. One such species is Vibrio cholerae, an autochthonous member of estuarine, riverine, and marine habitats and the causative agent of cholera, whose competence program is induced after colonization of chitin surfaces. In this study, we investigate how Vibrio cholerae's two extracellular nucleases, Xds and Dns, influence its natural transformability. We show that in the absence of Dns, transformation frequencies are significantly higher than in its presence. During growth on a chitin surface, an increase in transformation efficiency was found to correspond in time with increasing cell density and the repression of dns expression by the quorum-sensing regulator HapR. In contrast, at low cell density, the absence of HapR relieves dns repression, leading to the degradation of free DNA and to the abrogation of the transformation phenotype. Thus, as cell density increases, Vibrio cholerae undergoes a switch from nuclease-mediated degradation of extracellular DNA to the uptake of DNA by bacteria induced to a state of competence by chitin. Taken together, these results suggest the following model: nuclease production by low-density populations of V. cholerae might foster rapid growth by providing a source of nucleotides for the repletion of nucleotide pools. In contrast, the termination of nuclease production by static, high-density populations allows the uptake of intact DNA and coincides with a phase of potential genome diversification.

The Extracellular Nuclease Dns and Its Role in Natural Transformation of Vibrio cholerae▿

PubMed Central

Blokesch, Melanie; Schoolnik, Gary K.

2008-01-01

Free extracellular DNA is abundant in many aquatic environments. While much of this DNA will be degraded by nucleases secreted by the surrounding microbial community, some is available as transforming material that can be taken up by naturally competent bacteria. One such species is Vibrio cholerae, an autochthonous member of estuarine, riverine, and marine habitats and the causative agent of cholera, whose competence program is induced after colonization of chitin surfaces. In this study, we investigate how Vibrio cholerae's two extracellular nucleases, Xds and Dns, influence its natural transformability. We show that in the absence of Dns, transformation frequencies are significantly higher than in its presence. During growth on a chitin surface, an increase in transformation efficiency was found to correspond in time with increasing cell density and the repression of dns expression by the quorum-sensing regulator HapR. In contrast, at low cell density, the absence of HapR relieves dns repression, leading to the degradation of free DNA and to the abrogation of the transformation phenotype. Thus, as cell density increases, Vibrio cholerae undergoes a switch from nuclease-mediated degradation of extracellular DNA to the uptake of DNA by bacteria induced to a state of competence by chitin. Taken together, these results suggest the following model: nuclease production by low-density populations of V. cholerae might foster rapid growth by providing a source of nucleotides for the repletion of nucleotide pools. In contrast, the termination of nuclease production by static, high-density populations allows the uptake of intact DNA and coincides with a phase of potential genome diversification. PMID:18757542

[Isolation and purification of nonspecific nuclease of cyanobacterium Plectonema boryanum CALU 465].

PubMed

Tsymbal, N V; Samoĭlenko, V A; Syrchin, S A; Mendzhul, M I

2004-01-01

Nonspecific nuclease has been isolated from the cells of cyanobacterium Plectonema boryanum and purified to homogenic state. It has been established that the method of centrifugation of cell-free culture extract in the sucrose density gradient is efficient for the separation of pigment proteins and enzyme concentration. Under the successive use of two ion-exchangers the nuclease activity was determined in the concentration range of NaCl 0.065-0.085 M after separation of the cell-free cyanobacterium extract on the column with phosphocellulose in the range of 0.2-0.25 M, on the column with DEAE--Toyopearl respectively. The molecular mass of nuclease which is 40 kDa, has been determined by electrophoresis in polyacrylamide gel under denaturating conditions and gel-filtration on Sephadex G-100. It has been also established that the given enzyme is monosubunitary as to its structure.

Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae.

PubMed

Symington, L S; Kang, L E; Moreau, S

2000-12-01

A plasmid gap repair assay was used to assess the role of three known nucleases, Exo1, Mre11 and Rad1, in the processing of DNA ends and resolution of recombination intermediates during double-strand gap repair. In this assay, alterations in end processing or branch migration are reflected by the frequency of co-conversion of a chromosomal marker 200 bp from the gap. Gap repair associated with crossing over results in integration at the homologous chromosomal locus, whereas the plasmid remains episomal for non-crossover repair events. In mre11 strains, the frequency of gap repair was reduced 3- to 10-fold and conversion tracts were shorter than in the wild-type strain, consistent with a role for this nuclease in processing double-strand breaks. However, conversion tracts were longer in a strain containing the nuclease deficient allele, mre11-H125N, suggesting increased end processing by redundant nucleases. The frequency of gap repair was reduced 2-fold in rad1 mutants and crossing over was reduced, consistent with a role for Rad1 in cleaving recombination intermediates. The frequency of gap repair was increased in exo1 mutants with a significant increase in crossing over. In exo1 mre11 double mutants gap repair was reduced to below the mre11 single mutant level.

The SNM1B/APOLLO DNA nuclease functions in resolution of replication stress and maintenance of common fragile site stability.

PubMed

Mason, Jennifer M; Das, Ishita; Arlt, Martin; Patel, Neil; Kraftson, Stephanie; Glover, Thomas W; Sekiguchi, JoAnn M

2013-12-15

SNM1B/Apollo is a DNA nuclease that has important functions in telomere maintenance and repair of DNA interstrand crosslinks (ICLs) within the Fanconi anemia (FA) pathway. SNM1B is required for efficient localization of key repair proteins, such as the FA protein, FANCD2, to sites of ICL damage and functions epistatically to FANCD2 in cellular survival to ICLs and homology-directed repair. The FA pathway is also activated in response to replication fork stalling. Here, we sought to determine the importance of SNM1B in cellular responses to stalled forks in the absence of a blocking lesion, such as ICLs. We found that depletion of SNM1B results in hypersensitivity to aphidicolin, a DNA polymerase inhibitor that causes replication stress. We observed that the SNM1B nuclease is required for efficient localization of the DNA repair proteins, FANCD2 and BRCA1, to subnuclear foci upon aphidicolin treatment, thereby indicating SNM1B facilitates direct repair of stalled forks. Consistent with a role for SNM1B subsequent to recognition of the lesion, we found that SNM1B is dispensable for upstream events, including activation of ATR-dependent signaling and localization of RPA, γH2AX and the MRE11/RAD50/NBS1 complex to aphidicolin-induced foci. We determined that a major consequence of SNM1B depletion is a marked increase in spontaneous and aphidicolin-induced chromosomal gaps and breaks, including breakage at common fragile sites. Thus, this study provides evidence that SNM1B functions in resolving replication stress and preventing accumulation of genomic damage.

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

PubMed Central

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

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

PubMed

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.

Differential Trafficking of TLR1 I602S Underlies Host Protection Against Pathogenic Mycobacteria§

PubMed Central

Hart, Bryan E.; Tapping, Richard I.

2012-01-01

We have recently identified I602S as a frequent single nucleotide polymorphism of human TLR1 which greatly inhibits cell surface trafficking, confers hyporesponsiveness to TLR1 agonists, and protects against the mycobacterial diseases leprosy and tuberculosis. Since mycobacteria are known to manipulate the TLR system to their advantage, we hypothesize that the hyporesponsive 602S variant may confer protection by enabling the host to overcome this immune subversion. We report that primary human monocytes and macrophages from homozygous TLR1 602S individuals are resistant to mycobacterial-induced downregulation of macrophage MHCII, CD64, and IFNγ responses compared to individuals who harbor the TLR1 602I variant. Additionally, when challenged with mycobacterial agonists, macrophages from TLR1 602S/S individuals resist induction of host arginase-1; an enzyme that depletes cellular arginine stores required for production of antimicrobial reactive nitrogen intermediates. The differences in cell activation mediated by TLR1 602S and TLR1 602I are observed upon stimulation with soluble mycobacterial-derived agonists but not with whole mycobacterial cells. Taken together, these results suggest that the TLR1 602S variant protects against mycobacterial disease by preventing soluble mycobacterial products, perhaps released from granulomas, from disarming myeloid cells prior to their encounter with whole mycobacteria. PMID:23105135

Isolating DNA from sexual assault cases: a comparison of standard methods with a nuclease-based approach

PubMed Central

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

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

PubMed Central

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

Au nanoparticles/hollow molybdenum disulfide microcubes based biosensor for microRNA-21 detection coupled with duplex-specific nuclease and enzyme signal amplification.

PubMed

Shuai, Hong-Lei; Huang, Ke-Jing; Chen, Ying-Xu; Fang, Lin-Xia; Jia, Meng-Pei

2017-03-15

An ultrasensitive electrochemical biosensor for detecting microRNAs is fabricated based on hollow molybdenum disulfide (MoS 2 ) microcubes. Duplex-specific nuclease, enzyme and electrochemical-chemical-chemical redox cycling are used for signal amplification. Hollow MoS 2 microcubes constructed by ultrathin nanosheets are synthesized by a facile template-assisted strategy and used as supporting substrate. For biosensor assembling, biotinylated ssDNA capture probes are first immobilized on Au nanoparticles (AuNPs)/MoS 2 modified electrode in order to combine with streptavidin-conjugated alkaline phosphatase (SA-ALP). When capture probes hybridize with miRNAs, duplex-specific nuclease cleaves the formative duplexes. At the moment, the biotin group strips from the electrode surface and SA-ALP is incapacitated to attach onto electrode. Then, ascorbic acids induce the electrochemical-chemical-chemical redox cycling to produce electrochemical response in the presence of ferrocene methanol and tris (2-carboxyethyl) phosphine. Under optimum conditions, the proposed biosensor shows a good linear relationship between the current variation and logarithm of the microRNAs concentration ranging from 0.1fM to 0.1pM with a detection limit of 0.086fM (S/N=3). Furthermore, the biosensor is successfully applied to detect target miRNA-21 in human serum samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Installing logic gates in permeability controllable polyelectrolyte-carbon nitride films for detecting proteases and nucleases.

PubMed

Chen, Lichan; Zeng, Xiaoting; Dandapat, Anirban; Chi, Yuwu; Kim, Donghwan

2015-09-01

Proteases and nucleases are enzymes heavily involved in many important biological processes, such as cancer initiation, progression, and metastasis; hence, they are indicative of potential diagnostic biomarkers. Here, we demonstrate a new label free and sensitive electrochemiluminescent (ECL) sensing strategy for protease and nuclease assays that utilize target-triggered desorption of programmable polyelectrolyte films assembled on graphite-like carbon nitride (g-C3N4) film to regulate the diffusion flux of a coreactant. Furthermore, we have built Boolean logic gates OR and AND into the polyelectrolyte films, capable of simultaneously sensing proteases and nucleases in a complicated system by breaking it into simple functions. The developed intelligent permeability controlled enzyme sensor may prove valuable in future medical diagnostics.

Early zygote-specific nuclease in mitochondria of the true slime mold Physarum polycephalum.

PubMed

Moriyama, Yohsuke; Yamazaki, Tomokazu; Nomura, Hideo; Sasaki, Narie; Kawano, Shigeyuki

2005-11-01

The active, selective digestion of mtDNA from one parent is a possible molecular mechanism for the uniparental inheritance of mtDNA. In Physarum polycephalum, mtDNA is packed by DNA-binding protein Glom, which packs mtDNA into rod-shaped mt-nucleoids. After the mating, mtDNA from one parent is selectively digested, and the Glom began to disperse. Dispersed Glom was retained for at least 6 h after mtDNA digestion, but disappeared completely by about 12 h after mixing two strains. We identified two novel nucleases using DNA zymography with native-PAGE and SDS-PAGE. One is a Ca2+-dependent, high-molecular-weight nuclease complex (about 670 kDa), and the other is a Mn2+-dependent, high-molecular-weight nuclease complex (440-670 kDa); the activity of the latter was detected as a Mn2+-dependent, 13-kDa DNase band on SDS-PAGE. All mitochondria isolated from myxamoebae had mt-nucleoids, whereas half of the mitochondria isolated from the zygotes at 12 h after mixing had lost the mt-nucleoids. The activity of the Mn2+-dependent nuclease in the isolated mitochondria was detected at least 8 h after mixing of two strains. The timing and localization of the Mn2+-dependent DNase activity matched the selective digestion of mtDNA.

Functional analysis of Streptococcus pyogenes nuclease A (SpnA), a novel group A streptococcal virulence factor.

PubMed

Chang, Ann; Khemlani, Adrina; Kang, HaeJoo; Proft, Thomas

2011-03-01

Streptococcus pyogenes nuclease A (SpnA) is a recently discovered DNase that plays a role in virulence as shown in a mouse infection model. SpnA is the only cell wall-anchored DNase found in S. pyogenes thus far and shows a unique protein architecture. The C-terminal nuclease domain contains highly conserved catalytic site and Mg(2+) binding site residues. However, expression of the SpnA nuclease domain alone resulted in a soluble, but enzymatically inactive protein. We found that at least two out of three oligonucleotide/oligosaccharide-binding fold motifs found in the N-terminal domain are required for SpnA activity, probably contributing to substrate binding. Using a combination of a spnA deletion mutant and a Lactococcus lactis'gain-of-function' mutant, we have shown that SpnA promotes survival in whole human blood and in neutrophil killing assays and this is, at least in part, achieved by the destruction of neutrophil extracellular traps (NETs). We observed higher frequencies for anti-SpnA antibodies in streptococcal disease patient sera (79%, n = 19) compared with sera from healthy donors (33%, n = 9) suggesting that SpnA is expressed during infection. Detection of anti-SpnA antibodies in patient serum might be useful for the diagnostic of post-streptococcal diseases, such as acute rheumatic fever or glomerulonephritis. © 2011 Blackwell Publishing Ltd.

U1 small nuclear ribonucleoprotein particle-specific proteins interact with the first and second stem-loops of U1 RNA, with the A protein binding directly to the RNA independently of the 70K and Sm proteins.

PubMed Central

Patton, J R; Habets, W; van Venrooij, W J; Pederson, T

1989-01-01

The U1 small nuclear ribonucleoprotein particle (U1 snRNP), a cofactor in pre-mRNA splicing, contains three proteins, termed 70K, A, and C, that are not present in the other spliceosome-associated snRNPs. We studied the binding of the A and C proteins to U1 RNA, using a U1 snRNP reconstitution system and an antibody-induced nuclease protection technique. Antibodies that reacted with the A and C proteins induced nuclease protection of the first two stem-loops of U1 RNA in reconstituted U1 snRNP. Detailed analysis of the antibody-induced nuclease protection patterns indicated the existence of relatively long-range protein-protein interactions in the U1 snRNP, with the 5' end of U1 RNA and its associated specific proteins interacting with proteins bound to the Sm domain near the 3' end. UV cross-linking experiments in conjunction with an A-protein-specific antibody demonstrated that the A protein bound directly to the U1 RNA rather than assembling in the U1 snRNP exclusively via protein-protein interactions. This conclusion was supported by additional experiments revealing that the A protein could bind to U1 RNA in the absence of bound 70K and Sm core proteins. Images PMID:2529425

The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities.

PubMed

Allerston, Charles K; Lee, Sook Y; Newman, Joseph A; Schofield, Christopher J; McHugh, Peter J; Gileadi, Opher

2015-12-15

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-β-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-β-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

PubMed Central

2014-01-01

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

S-Glutathionylation of Keap1: a new role for glutathione S-transferase pi in neuronal protection.

PubMed

Carvalho, Andreia Neves; Marques, Carla; Guedes, Rita C; Castro-Caldas, Margarida; Rodrigues, Elsa; van Horssen, Jack; Gama, Maria João

2016-05-01

Oxidative stress is a key pathological feature of Parkinson's disease (PD). Glutathione S-transferase pi (GSTP) is a neuroprotective antioxidant enzyme regulated at the transcriptional level by the antioxidant master regulator nuclear factor-erythroid 2-related factor 2 (Nrf2). Here, we show for the first time that upon MPTP-induced oxidative stress, GSTP potentiates S-glutathionylation of Kelch-like ECH-associated protein 1 (Keap1), an endogenous repressor of Nrf2, in vivo. S-glutathionylation of Keap1 leads to Nrf2 activation and subsequently increases expression of GSTP. This positive feedback regulatory loop represents a novel mechanism by which GSTP elicits antioxidant protection in the brain. © 2016 Federation of European Biochemical Societies.

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

PubMed

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.

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

PubMed

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.

Crystal structures of the structure-selective nuclease Mus81-Eme1 bound to flap DNA substrates

PubMed Central

Gwon, Gwang Hyeon; Jo, Aera; Baek, Kyuwon; Jin, Kyeong Sik; Fu, Yaoyao; Lee, Jong-Bong; Kim, YoungChang; Cho, Yunje

2014-01-01

The Mus81-Eme1 complex is a structure-selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross-links, replication fork collapse, or double-strand breaks. To explain the molecular basis of 3′ flap substrate recognition and cleavage mechanism by Mus81-Eme1, we determined crystal structures of human Mus81-Eme1 bound to various flap DNA substrates. Mus81-Eme1 undergoes gross substrate-induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre- and post-nick duplex DNA and (ii) a “5′ end binding pocket” that hosts the 5′ nicked end of post-nick DNA. These features are crucial for comprehensive protein-DNA interaction, sharp bending of the 3′ flap DNA substrate, and incision strand placement at the active site. While Mus81-Eme1 unexpectedly shares several common features with members of the 5′ flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81-Eme1 preferentially cleaves 3′ flap DNA substrates with 5′ nicked ends. PMID:24733841

Proline cis-trans isomerization in staphylococcal nuclease: multi-substrate free energy perturbation calculations.

PubMed Central

Hodel, A.; Rice, L. M.; Simonson, T.; Fox, R. O.; Brünger, A. T.

1995-01-01

Staphylococcal nuclease A exists in two folded forms that differ in the isomerization state of the Lys 116-Pro 117 peptide bond. The dominant form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small difference in free energy between the cis and trans forms (delta Gcis-->trans approximately 1.2 kcal/mol) results from large and nearly compensating differences in enthalpy and entropy (delta Hcis-->trans approximately delta TScis-->trans approximately 10 kcal/mol). There is evidence from X-ray crystal structures that the structural differences between the cis and the trans forms of nuclease are confined to the conformation of residues 112-117, a solvated protein loop. Here, we obtain a thermodynamic and structural description of the conformational equilibrium of this protein loop through an exhaustive conformational search that identified several substates followed by free energy simulations between the substrates. By partitioning the search into conformational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy values. The protein and water environment was implicitly modeled by appropriately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a small free energy difference between the cis and trans forms composed of larger, compensating differences in enthalpy and entropy. The structural predictions of these simulations were qualitatively consistent with known X-ray structures of nuclease variants and yield a model of the unknown minor trans conformation. PMID:7613463

A Novel Nuclease Activity that is Activated by Ca2+ Chelated to EGTA

PubMed Central

Dominguez, Kenneth; Ward, W. Steven

2010-01-01

Most nucleases require a divalent cation as a cofactor, usually Mg2+ or Ca2+, and are inhibited by the chelators EDTA and EGTA. We report the existence of a novel nuclease activity, initially identified in the luminal fluids of the mouse male reproductive tract but subsequently found in other tissues, that requires EGTA chelated to calcium to digest DNA. We refer to this unique enzyme as CEAN (Chelated EGTA Activated Nuclease). Using a fraction of vas deferens luminal fluid, plasmid DNA was degraded in the presence of excess Ca2+ (Ca2+:EGTA = 16) or excess EGTA (Ca2+:EGTA = 0.25), but required the presence of both. Higher levels of EGTA (Ca2+:EGTA = 0.10) prevented activity, suggesting that unchelated EGTA may be a competitive inhibitor. The EGTA-Ca2+ activation of CEAN is reversible as removing EGTA-Ca2+ stops ongoing DNA degradation, but adding EGTA-Ca2+ again reactivates the enzyme. This suggests the possibility that CEAN binds directly to EGTA-Ca2+. CEAN has a greater specificity for the chelator than for the divalent cation. Two other chelators, BAPTA and sodium citrate, do not activate CEAN in the presence of cation, but chelated EDTA does. EGTA chelated to other divalent cations such as Mn2+, Zn2+, and Cu2+ activate CEAN, but not Mg2+. The activity is lost upon boiling suggesting that it is a protein. These data suggest that EGTA and EDTA may not always prevent DNA from nuclease damage. PMID:19938954

Generation of Esr1-Knockout Rats Using Zinc Finger Nuclease-Mediated Genome Editing

PubMed Central

Dhakal, Pramod; Kubota, Kaiyu; Chakraborty, Damayanti; Lei, Tianhua; Larson, Melissa A.; Wolfe, Michael W.; Roby, Katherine F.; Vivian, Jay L.

2014-01-01

Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17β-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action. PMID:24506075

Generation of Esr1-knockout rats using zinc finger nuclease-mediated genome editing.

PubMed

Rumi, M A Karim; Dhakal, Pramod; Kubota, Kaiyu; Chakraborty, Damayanti; Lei, Tianhua; Larson, Melissa A; Wolfe, Michael W; Roby, Katherine F; Vivian, Jay L; Soares, Michael J

2014-05-01

Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17β-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action.

Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

PubMed Central

Caljon, Guy; Ridder, Karin De; Stijlemans, Benoît; Coosemans, Marc; Magez, Stefan; De Baetselier, Patrick; Van Den Abbeele, Jan

2012-01-01

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 KD 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. PMID:23110062

Electrostatic effects in unfolded staphylococcal nuclease

PubMed Central

Fitzkee, Nicholas C.; García-Moreno E, Bertrand

2008-01-01

Structure-based calculations of pK a values and electrostatic free energies of proteins assume that electrostatic effects in the unfolded state are negligible. In light of experimental evidence showing that this assumption is invalid for many proteins, and with increasing awareness that the unfolded state is more structured and compact than previously thought, a detailed examination of electrostatic effects in unfolded proteins is warranted. Here we address this issue with structure-based calculations of electrostatic interactions in unfolded staphylococcal nuclease. The approach involves the generation of ensembles of structures representing the unfolded state, and calculation of Coulomb energies to Boltzmann weight the unfolded state ensembles. Four different structural models of the unfolded state were tested. Experimental proton binding data measured with a variant of nuclease that is unfolded under native conditions were used to establish the validity of the calculations. These calculations suggest that weak Coulomb interactions are an unavoidable property of unfolded proteins. At neutral pH, the interactions are too weak to organize the unfolded state; however, at extreme pH values, where the protein has a significant net charge, the combined action of a large number of weak repulsive interactions can lead to the expansion of the unfolded state. The calculated pK a values of ionizable groups in the unfolded state are similar but not identical to the values in small peptides in water. These studies suggest that the accuracy of structure-based calculations of electrostatic contributions to stability cannot be improved unless electrostatic effects in the unfolded state are calculated explicitly. PMID:18227429

Genome Editing in Mice Using TALE Nucleases.

PubMed

Wefers, Benedikt; Brandl, Christina; Ortiz, Oskar; Wurst, Wolfgang; Kühn, Ralf

2016-01-01

Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.

The antigenic surface of staphylococcal nuclease. II. Analysis of the N-1 epitope by site-directed mutagenesis.

PubMed

Smith, A M; Benjamin, D C

1991-02-15

Previous studies in our laboratory on the production and isolation of a panel of mAb to staphylococcal nuclease allowed us to define a series of eight overlapping epitopes. Using site-directed mutagenesis of the nuclease coding sequences we were able to map the nonoverlapping epitopes recognized by two members of this panel. In the study reported here, we report the generation and analysis of a number of single amino acid substitutions for seven surface residues predicted to lie within one of these two epitopes. Immunochemical analysis showed that one or more substitutions at each of these seven positions had a major effect on mAb binding, whereas other substitutions had none. Based on the nature of these substitutions and the chemical and physical properties of the variant molecules, we believe that any structural effects induced by these substitutions are local and do not result in long-range structural alterations that indirectly influence antibody reactivity. Therefore, we conclude that disruption of mAb binding can be directly attributed to changes in amino acid side chains and that not only are all seven of the residues studied part of the epitope but all seven make contact with the antibody combining site. These studies demonstrate the advantages of using site-directed mutagenesis to study antigen structure and emphasize the importance of constructing the examining multiple substitutions for any given amino acid.

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

PubMed

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.

Detection of single-nucleotide polymorphisms using gold nanoparticles and single-strand-specific nucleases.

PubMed

Chen, Yen-Ting; Hsu, Chiao-Ling; Hou, Shao-Yi

2008-04-15

The current study reports an assay approach that can detect single-nucleotide polymorphisms (SNPs) and identify the position of the point mutation through a single-strand-specific nuclease reaction and a gold nanoparticle assembly. The assay can be implemented via three steps: a single-strand-specific nuclease reaction that allows the enzyme to truncate the mutant DNA; a purification step that uses capture probe-gold nanoparticles and centrifugation; and a hybridization reaction that induces detector probe-gold nanoparticles, capture probe-gold nanoparticles, and the target DNA to form large DNA-linked three-dimensional aggregates of gold nanoparticles. At high temperature (63 degrees C in the current case), the purple color of the perfect match solution would not change to red, whereas a mismatched solution becomes red as the assembled gold nanoparticles separate. Using melting analysis, the position of the point mutation could be identified. This assay provides a convenient colorimetric detection that enables point mutation identification without the need for expensive mass spectrometry. To our knowledge, this is the first report concerning SNP detection based on a single-strand-specific nuclease reaction and a gold nanoparticle assembly.

(NZ)CH...O contacts assist crystallization of a ParB-like nuclease.

PubMed

Shaw, Neil; Cheng, Chongyun; Tempel, Wolfram; Chang, Jessie; Ng, Joseph; Wang, Xin-Yu; Perrett, Sarah; Rose, John; Rao, Zihe; Wang, Bi-Cheng; Liu, Zhi-Jie

2007-07-07

The major bottleneck for determination of 3 D structures of proteins using X-rays is the production of diffraction quality crystals. Often proteins are subjected to chemical modification to improve the chances of crystallization Here, we report the successful crystallization of a nuclease employing a reductive methylation protocol. The key to crystallization was the successful introduction of 44 new cohesive (NZ) CH...O contacts (3.2-3.7 A) by the addition of 2 methyl groups to the side chain amine nitrogen (NZ) of 9 lysine residues of the nuclease. The new contacts dramatically altered the crystallization properties of the protein, resulting in crystals that diffracted to 1.2 A resolution. Analytical ultracentrifugation analysis and thermodynamics results revealed a more compact protein structure with better solvent exclusion of buried Trp residues in the folded state of the methylated protein, assisting crystallization. In this study, introduction of novel cohesive (NZ)CH...O contacts by reductive methylation resulted in the crystallization of a protein that had previously resisted crystallization in spite of extensive purification and crystallization space screening. Introduction of (NZ)CH...O contacts could provide a solution to crystallization problems for a broad range of protein targets.

Mouse Spermatozoa Contain a Nuclease that Is Activated by Pretreatment with EGTA and Subsequent Calcium Incubation

PubMed Central

Boaz, Segal M.; Dominguez, Kenneth; Shaman, Jeffrey A.; Ward, W. Steven

2009-01-01

We demonstrated that mouse spermatozoa cleave their DNA into ~50 kb loop-sized fragments with topoisomerase IIB when treated with MnCl2 and CaCl2 in a process we term sperm chromatin fragmentation (SCF). SCF can be reversed by EDTA. A nuclease then further degrades the DNA in a process we term sperm DNA degradation (SDD). MnCl2 alone could elicit this activity, but CaCl2 had no effect. Here, we demonstrate the existence of a nuclease in the vas deferens that can be activated by EGTA to digest the sperm DNA by SDD. Spermatozoa were extracted with salt and dithiothreitol to remove protamines and then incubated with EGTA. Next, the EGTA was removed and divalent cations were added. We found that Mn+2, Ca+2, or Zn+2 could each activate SDD in spermatozoa but Mg+2 could not. When the reaction was slowed by incubation on ice, EGTA pretreatment followed by incubation in Ca+2 elicited the reversible fragmentation of sperm DNA evident in SCF. When the reactions were then incubated at 37°C they progressed to the more complete degradation of DNA by SDD. EDTA could also be used to activate the nuclease, but required a higher concentration than EGTA. This EGTA-activatable nuclease activity was found in each fraction of the vas deferens plasma: in the spermatozoa, in the surrounding fluid, and in the insoluble components in the fluid. These results suggest that this sperm nuclease is regulated by a mechanism that is sensitive to EGTA, possibly by removing inhibition of a calcium binding protein. PMID:17879959

GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases

PubMed Central

Nguyen, Nhu T.; Liebers, Matthew; Topkar, Ved V.; Thapar, Vishal; Wyvekens, Nicolas; Khayter, Cyd; Iafrate, A. John; Le, Long P.; Aryee, Martin J.; Joung, J. Keith

2014-01-01

CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called Genome-wide Unbiased Identification of DSBs Enabled by Sequencing (GUIDE-Seq), relies on capture of double-stranded oligodeoxynucleotides into breaks Application of GUIDE-Seq to thirteen RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or ChIP-Seq. GUIDE-Seq also identified RGN-independent genomic breakpoint ‘hotspots’. Finally, GUIDE-Seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases prior to clinical use. PMID:25513782

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease.

PubMed

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.

Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease.

PubMed

Liu, Yibin; Song, Chen; Ladas, Ioannis; Fitarelli-Kiehl, Mariana; Makrigiorgos, G Mike

2017-04-07

Aberrant methylation changes, often present in a minor allelic fraction in clinical samples such as plasma-circulating DNA (cfDNA), are potentially powerful prognostic and predictive biomarkers in human disease including cancer. We report on a novel, highly-multiplexed approach to facilitate analysis of clinically useful methylation changes in minor DNA populations. Methylation Specific Nuclease-assisted Minor-allele Enrichment (MS-NaME) employs a double-strand-specific DNA nuclease (DSN) to remove excess DNA with normal methylation patterns. The technique utilizes oligonucleotide-probes that direct DSN activity to multiple targets in bisulfite-treated DNA, simultaneously. Oligonucleotide probes targeting unmethylated sequences generate local double stranded regions resulting to digestion of unmethylated targets, and leaving methylated targets intact; and vice versa. Subsequent amplification of the targeted regions results in enrichment of the targeted methylated or unmethylated minority-epigenetic-alleles. We validate MS-NaME by demonstrating enrichment of RARb2, ATM, MGMT and GSTP1 promoters in multiplexed MS-NaME reactions (177-plex) using dilutions of methylated/unmethylated DNA and in DNA from clinical lung cancer samples and matched normal tissue. MS-NaME is a highly scalable single-step approach performed at the genomic DNA level in solution that combines with most downstream detection technologies including Sanger sequencing, methylation-sensitive-high-resolution melting (MS-HRM) and methylation-specific-Taqman-based-digital-PCR (digital Methylight) to boost detection of low-level aberrant methylation-changes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template.

PubMed

Sather, Blythe D; Romano Ibarra, Guillermo S; Sommer, Karen; Curinga, Gabrielle; Hale, Malika; Khan, Iram F; Singh, Swati; Song, Yumei; Gwiazda, Kamila; Sahni, Jaya; Jarjour, Jordan; Astrakhan, Alexander; Wagner, Thor A; Scharenberg, Andrew M; Rawlings, David J

2015-09-30

Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties. Copyright © 2015, American Association for the Advancement of Science.

megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.

PubMed

Boissel, Sandrine; Jarjour, Jordan; Astrakhan, Alexander; Adey, Andrew; Gouble, Agnès; Duchateau, Philippe; Shendure, Jay; Stoddard, Barry L; Certo, Michael T; Baker, David; Scharenberg, Andrew M

2014-02-01

Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at 'off-target' sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate 'megaTAL', in which the DNA binding region of a transcription activator-like (TAL) effector is used to 'address' a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.

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

PubMed Central

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

Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein–staphylococcal nuclease hybrid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mori, Tomoaki; Nakamura, Kento; Masaoka, Keisuke

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 constructmore » 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.« less

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

PubMed

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.

Loop propensity of the sequence YKGQP from staphylococcal nuclease: implications for the folding of nuclease.

PubMed

Patel, Sunita; Sasidhar, Yellamraju U

2007-10-01

Recently we performed molecular dynamics (MD) simulations on the folding of the hairpin peptide DTVKLMYKGQPMTFR from staphylococcal nuclease in explicit water. We found that the peptide folds into a hairpin conformation with native and nonnative hydrogen-bonding patterns. In all the folding events observed in the folding of the hairpin peptide, loop formation involving the region YKGQP was an important event. In order to trace the origins of the loop propensity of the sequence YKGQP, we performed MD simulations on the sequence starting from extended, polyproline II and native type I' turn conformations for a total simulation length of 300 ns, using the GROMOS96 force field under constant volume and temperature (NVT) conditions. The free-energy landscape of the peptide YKGQP shows minima corresponding to loop conformation with Tyr and Pro side-chain association, turn and extended conformational forms, with modest free-energy barriers separating the minima. To elucidate the role of Gly in facilitating loop formation, we also performed MD simulations of the mutated peptide YKAQP (Gly --> Ala mutation) under similar conditions starting from polyproline II conformation for 100 ns. Two minima corresponding to bend/turn and extended conformations were observed in the free-energy landscape for the peptide YKAQP. The free-energy barrier between the minima in the free-energy landscape of the peptide YKAQP was also modest. Loop conformation is largely sampled by the YKGQP peptide, while extended conformation is largely sampled by the YKAQP peptide. We also explain why the YKGQP sequence samples type II turn conformation in these simulations, whereas the sequence as part of the hairpin peptide DTVKLMYKGQPMTFR samples type I' turn conformation both in the X-ray crystal structure and in our earlier simulations on the folding of the hairpin peptide. We discuss the implications of our results to the folding of the staphylococcal nuclease. Copyright (c) 2007 European Peptide

Structural insights into 5‧ flap DNA unwinding and incision by the human FAN1 dimer

NASA Astrophysics Data System (ADS)

Zhao, Qi; Xue, Xiaoyu; Longerich, Simonne; Sung, Patrick; Xiong, Yong

2014-12-01

Human FANCD2-associated nuclease 1 (FAN1) is a DNA structure-specific nuclease involved in the processing of DNA interstrand crosslinks (ICLs). FAN1 maintains genomic stability and prevents tissue decline in multiple organs, yet it confers ICL-induced anti-cancer drug resistance in several cancer subtypes. Here we report three crystal structures of human FAN1 in complex with a 5‧ flap DNA substrate, showing that two FAN1 molecules form a head-to-tail dimer to locate the lesion, orient the DNA and unwind a 5‧ flap for subsequent incision. Biochemical experiments further validate our model for FAN1 action, as structure-informed mutations that disrupt protein dimerization, substrate orientation or flap unwinding impair the structure-specific nuclease activity. Our work elucidates essential aspects of FAN1-DNA lesion recognition and a unique mechanism of incision. These structural insights shed light on the cellular mechanisms underlying organ degeneration protection and cancer drug resistance mediated by FAN1.

Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing.

PubMed

Miyaoka, Yuichiro; Berman, Jennifer R; Cooper, Samantha B; Mayerl, Steven J; Chan, Amanda H; Zhang, Bin; Karlin-Neumann, George A; Conklin, Bruce R

2016-03-31

Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR-based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator-like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.

Functionalized bioengineered spider silk spheres improve nuclease resistance and activity of oligonucleotide therapeutics providing a strategy for cancer treatment.

PubMed

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

Inhibition of miR-128-3p by Tongxinluo Protects Human Cardiomyocytes from Ischemia/reperfusion Injury via Upregulation of p70s6k1/p-p70s6k1

PubMed Central

Chen, Gui-hao; Xu, Chuan-sheng; Zhang, Jie; Li, Qing; Cui, He-he; Li, Xiang-dong; Chang, Li-ping; Tang, Rui-jie; Xu, Jun-yan; Tian, Xia-qiu; Huang, Pei-sen; Xu, Jun; Jin, Chen; Yang, Yue-jin

2017-01-01

Background and Aims: Tongxinluo (TXL) is a multifunctional traditional Chinese medicine that has been widely used to treat cardiovascular and cerebrovascular diseases. However, no studies have explored whether TXL can protect human cardiomyocytes (HCMs) from ischemia/reperfusion (I/R) injury. Reperfusion Injury Salvage Kinase (RISK) pathway activation was previously demonstrated to protect the hearts against I/R injury and it is generally activated via Akt or (and) Erk 1/2, and their common downstream protein, ribosomal protein S6 kinase (p70s6k). In addition, prior studies proved that TXL treatment of cells promoted secretion of VEGF, which could be stimulated by the increased phosphorylation of one p70s6k subtype, p70s6k1. Consequently, we hypothesized TXL could protect HCMs from I/R injury by activating p70s6k1 and investigated the underlying mechanism. Methods and Results: HCMs were exposed to hypoxia (18 h) and reoxygenation (2 h) (H/R), with or without TXL pretreatment. H/R reduced mitochondrial membrane potential, increased bax/bcl-2 ratios and cytochrome C levels and induced HCM apoptosis. TXL preconditioning reversed these H/R-induced changes in a dose-dependent manner and was most effective at 400 μg/mL. The anti-apoptotic effect of TXL was abrogated by rapamycin, an inhibitor of p70s6k. However, inhibitors of Erk1/2 (U0126) or Akt (LY294002) failed to inhibit the protective effect of TXL. TXL increased p70s6k1 expression and, thus, enhanced its phosphorylation. Furthermore, transfection of cardiomyocytes with siRNA to p70s6k1 abolished the protective effects of TXL. Among the micro-RNAs (miR-145-5p, miR-128-3p and miR-497-5p) previously reported to target p70s6k1, TXL downregulated miR-128-3p in HCMs during H/R, but had no effects on miR-145-5p and miR-497-5p. An in vivo study confirmed the role of the p70s6k1 pathway in the infarct-sparing effect of TXL, demonstrating that TXL decreased miR-128-3p levels in the rat myocardium during I/R. Transfection

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

PubMed

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.

Cloning and Expression of the Erwinia carotovora subsp. carotovora Gene Encoding the Low-Molecular-Weight Bacteriocin Carocin S1▿

PubMed Central

Chuang, Duen-yau; Chien, Yung-chei; Wu, Huang-Pin

2007-01-01

The purpose of this study was to clone the carocin S1 gene and express it in a non-carocin-producing strain of Erwinia carotovora. A mutant, TH22-10, which produced a high-molecular-weight bacteriocin but not a low-molecular-weight bacteriocin, was obtained by Tn5 insertional mutagenesis using H-rif-8-2 (a spontaneous rifampin-resistant mutant of Erwinia carotovora subsp. carotovora 89-H-4). Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of the contiguous 2,280-bp region were determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequence fragment. ORF2 and ORF3 were identified with the carocin S1 genes, caroS1K (ORF2) and caroS1I (ORF3), which, respectively, encode a killing protein (CaroS1K) and an immunity protein (CaroS1I). These genes were homologous to the pyocin S3 gene and the pyocin AP41 gene. Carocin S1 was expressed in E. carotovora subsp. carotovora Ea1068 and replicated in TH22-10 but could not be expressed in Escherichia coli (JM101) because a consensus sequence resembling an SOS box was absent. A putative sequence similar to the consensus sequence for the E. coli cyclic AMP receptor protein binding site (−312 bp) was found upstream of the start codon. Production of this bacteriocin was also induced by glucose and lactose. The homology search results indicated that the carocin S1 gene (between bp 1078 and bp 1704) was homologous to the pyocin S3 and pyocin AP41 genes in Pseudomonas aeruginosa. These genes encode proteins with nuclease activity (domain 4). This study found that carocin S1 also has nuclease activity. PMID:17071754

Design of nuclease-based target recycling signal amplification in aptasensors.

PubMed

Yan, Mengmeng; Bai, Wenhui; Zhu, Chao; Huang, Yafei; Yan, Jiao; Chen, Ailiang

2016-03-15

Compared with conventional antibody-based immunoassay methods, aptasensors based on nucleic acid aptamer have made at least two significant breakthroughs. One is that aptamers are more easily used for developing various simple and rapid homogeneous detection methods by "sample in signal out" without multi-step washing. The other is that aptamers are more easily employed for developing highly sensitive detection methods by using various nucleic acid-based signal amplification approaches. As many substances playing regulatory roles in physiology or pathology exist at an extremely low concentration and many chemical contaminants occur in trace amounts in food or environment, aptasensors for signal amplification contribute greatly to detection of such targets. Among the signal amplification approaches in highly sensitive aptasensors, the nuclease-based target recycling signal amplification has recently become a research focus because it shows easy design, simple operation, and rapid reaction and can be easily developed for homogenous assay. In this review, we summarized recent advances in the development of various nuclease-based target recycling signal amplification with the aim to provide a general guide for the design of aptamer-based ultrasensitive biosensing assays. Copyright © 2015 Elsevier B.V. All rights reserved.

Genome Editing in Mouse Spermatogonial Stem/Progenitor Cells Using Engineered Nucleases

PubMed Central

Fanslow, Danielle A.; Wirt, Stacey E.; Barker, Jenny C.; Connelly, Jon P.; Porteus, Matthew H.; Dann, Christina Tenenhaus

2014-01-01

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. PMID:25409432

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

PubMed

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

Nanoplasmonic molecular ruler for nuclease activity and DNA footprinting

DOEpatents

Chen, Fanqing Frank; Liu, Gang L; Lee, Luke P

2013-10-29

This invention provides a nanoplasmonic molecular ruler, which can perform label-free and real-time monitoring of nucleic acid (e.g., DNA) length changes and perform nucleic acid footprinting. In various embodiments the ruler comprises a nucleic acid attached to a nanoparticle, such that changes in the nucleic acid length are detectable using surface plasmon resonance. The nanoplasmonic ruler provides a fast and convenient platform for mapping nucleic acid-protein interactions, for nuclease activity monitoring, and for other footprinting related methods.

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

DTIC Science & Technology

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

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-03-01

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

Zinc-finger nucleases-based genome engineering to generate isogenic human cell lines.

PubMed

Dreyer, Anne-Kathrin; Cathomen, Toni

2012-01-01

Customized zinc-finger nucleases (ZFNs) have developed into a promising technology to precisely alter mammalian genomes for biomedical research, biotechnology, or human gene therapy. In the context of synthetic biology, the targeted integration of a transgene or reporter cassette into a "neutral site" of the human genome, such as the AAVS1 locus, permits the generation of isogenic human cell lines with two major advantages over standard genetic manipulation techniques: minimal integration site-dependent effects on the transgene and, vice versa, no functional perturbation of the host-cell transcriptome. Here we describe in detail how ZFNs can be employed to target integration of a transgene cassette into the AAVS1 locus and how to characterize the targeted cells by PCR-based genotyping.

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

PubMed Central

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

Liposomal SLA co-incorporated with PO CpG ODNs or PS CpG ODNs induce the same protection against the murine model of leishmaniasis.

PubMed

Shargh, Vahid Heravi; Jaafari, Mahmoud Reza; Khamesipour, Ali; Jaafari, Iman; Jalali, Seyed Amir; Abbasi, Azam; Badiee, Ali

2012-06-06

First generation Leishmania vaccines consisting of whole killed parasites with or without adjuvants have reached phase 3 trial and failed to show enough efficacy mainly due to the lack of an appropriate adjuvant. In this study, the nuclease-resistant phosphorothioate CpG oligodeoxynucleotides (PS CpG) or nuclease-sensitive phosphodiester CpG ODNs (PO CpG) were used as adjuvants to enhance immunogenicity and rate of protection against leishmaniasis. Due to the susceptibility of PO CpG to nuclease degradation, an efficient liposomal delivery system was developed to protect them from degradation. 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid was used because of its unique adjuvanticity and electrostatic interaction with negatively charged CpG ODNs. To evaluate the role of liposomal formulation in protection rate and enhanced immune response, BALB/c mice were immunized subcutaneously with liposomal soluble Leishmania antigens (SLA) co-incorporated with PO CpG (Lip-SLA-PO CpG), Lip-SLA-PS CpG, SLA+PO CpG, SLA+PS CpG, SLA or buffer. As criteria for protection, footpad swelling at the site of challenge, parasite loads, the levels of IFN-γ and IL-4, and the IgG subtypes were evaluated. The groups of mice receiving Lip-SLA-PO CpG or Lip-SLA-PS CpG showed a high protection rate compared with the control groups. In addition, there was no significant difference in immune response generation between mice immunized with PS CpG and the group receiving PO CpG when incorporated into the liposomes. The results suggested that liposomal form of PO CpG might be used instead of PS CpG in future vaccine formulations as an efficient adjuvant. Copyright © 2012 Elsevier Ltd. All rights reserved.

Two Decades of Enhancing Children’s Environmental Health Protection at the U.S. Environmental Protection Agency

PubMed Central

Firestone, Michael; Berger, Martha; Foos, Brenda; Etzel, Ruth

2016-01-01

Summary: This article provides an overview of public health efforts by the U.S. Environmental Protection Agency (EPA) during the past two decades to protect children’s health from environmental hazards. It highlights examples of concrete steps and accomplishments toward improving environmental protection and health outcomes achieved through public policy, rules and regulations, increased scientific understanding, and public health messaging. Additionally, examples of future challenges for better understanding and improving children’s environmental health are discussed. PMID:27905272

The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities

PubMed Central

Allerston, Charles K.; Lee, Sook Y.; Newman, Joseph A.; Schofield, Christopher J.; McHugh, Peter J.; Gileadi, Opher

2015-01-01

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-β-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-β-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions. PMID:26582912

Roles of SLX1–SLX4, MUS81–EME1, and GEN1 in avoiding genome instability and mitotic catastrophe

PubMed Central

Sarbajna, Shriparna; Davies, Derek; West, Stephen C.

2014-01-01

The resolution of recombination intermediates containing Holliday junctions (HJs) is critical for genome maintenance and proper chromosome segregation. Three pathways for HJ processing exist in human cells and involve the following enzymes/complexes: BLM–TopoIIIα–RMI1–RMI2 (BTR complex), SLX1–SLX4–MUS81–EME1 (SLX–MUS complex), and GEN1. Cycling cells preferentially use the BTR complex for the removal of double HJs in S phase, with SLX–MUS and GEN1 acting at temporally distinct phases of the cell cycle. Cells lacking SLX–MUS and GEN1 exhibit chromosome missegregation, micronucleus formation, and elevated levels of 53BP1-positive G1 nuclear bodies, suggesting that defects in chromosome segregation lead to the transmission of extensive DNA damage to daughter cells. In addition, however, we found that the effects of SLX4, MUS81, and GEN1 depletion extend beyond mitosis, since genome instability is observed throughout all phases of the cell cycle. This is exemplified in the form of impaired replication fork movement and S-phase progression, endogenous checkpoint activation, chromosome segmentation, and multinucleation. In contrast to SLX4, SLX1, the nuclease subunit of the SLX1–SLX4 structure-selective nuclease, plays no role in the replication-related phenotypes associated with SLX4/MUS81 and GEN1 depletion. These observations demonstrate that the SLX1–SLX4 nuclease and the SLX4 scaffold play divergent roles in the maintenance of genome integrity in human cells. PMID:24831703

Recombinant Newcastle disease virus expressing the infectious bronchitis virus S1 gene protects chickens against Newcastle disease virus and infectious bronchitis virus challenge.

PubMed

Zhao, Ran; Sun, Junfeng; Qi, Tianming; Zhao, Wen; Han, Zongxi; Yang, Xiaopu; Liu, Shengwang

2017-04-25

The recombinant LaSota strain expressing a chimeric IBV S1 gene (rLaSota-S1) was constructed with the S1 gene of the LX4 type IBV ck/CH/LDL/091022. The expression of the S1 protein was detected by an indirect immunofluorescence assay and Western blotting. The rLaSota-S1 strain was slightly attenuated, and its growth dynamics were similar to that of the parental LaSota strain. Vaccination of specific pathogen-free chickens with the rLaSota-S1 strain induced NDV hemagglutination inhibition antibodies, and it protected chickens from challenge with virulent NDV. In addition, vaccination with the rLaSota-S1 strain induced IBV-specific IgG antibodies and cellular immunity; however, a single vaccination provided partial protection with reduced virus shedding. Better protection efficiency was observed after a booster vaccination, which resulted in higher antibody titers, significantly fewer disease symptoms, and reduced virus replication and shedding. Our results suggest that the rLaSota-S1 strain is a bivalent vaccine candidate against both NDV and IBV. Copyright © 2017 Elsevier Ltd. All rights reserved.

Gene Editing in Human Lymphoid Cells: Role for Donor DNA, Type of Genomic Nuclease and Cell Selection Method.

PubMed

Zotova, Anastasia; Lopatukhina, Elena; Filatov, Alexander; Khaitov, Musa; Mazurov, Dmitriy

2017-11-02

Programmable endonucleases introduce DNA breaks at specific sites, which are repaired by non-homologous end joining (NHEJ) or homology recombination (HDR). Genome editing in human lymphoid cells is challenging as these difficult-to-transfect cells may also inefficiently repair DNA by HDR. Here, we estimated efficiencies and dynamics of knockout (KO) and knockin (KI) generation in human T and B cell lines depending on repair template, target loci and types of genomic endonucleases. Using zinc finger nuclease (ZFN), we have engineered Jurkat and CEM cells with the 8.2 kb human immunodeficiency virus type 1 (HIV-1) ∆Env genome integrated at the adeno-associated virus integration site 1 (AAVS1) locus that stably produce virus particles and mediate infection upon transfection with helper vectors. Knockouts generated by ZFN or clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) double nicking techniques were comparably efficient in lymphoid cells. However, unlike polyclonal sorted cells, gene-edited cells selected by cloning exerted tremendous deviations in functionality as estimated by replication of HIV-1 and human T cell leukemia virus type 1 (HTLV-1) in these cells. Notably, the recently reported high-fidelity eCas9 1.1 when combined to the nickase mutation displayed gene-dependent decrease in on-target activity. Thus, the balance between off-target effects and on-target efficiency of nucleases, as well as choice of the optimal method of edited cell selection should be taken into account for proper gene function validation in lymphoid cells.

Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation

PubMed Central

Seper, Andrea; Fengler, Vera H I; Roier, Sandro; Wolinski, Heimo; Kohlwein, Sepp D; Bishop, Anne L; Camilli, Andrew; Reidl, Joachim; Schild, Stefan

2011-01-01

Biofilms are a preferred mode of survival for many microorganisms including Vibrio cholerae, the causative agent of the severe secretory diarrhoeal disease cholera. The ability of the facultative human pathogen V. cholerae to form biofilms is a key factor for persistence in aquatic ecosystems and biofilms act as a source for new outbreaks. Thus, a better understanding of biofilm formation and transmission of V. cholerae is an important target to control the disease. So far the Vibrio exopolysaccharide was the only known constituent of the biofilm matrix. In this study we identify and characterize extracellular DNA as a component of the Vibrio biofilm matrix. Furthermore, we show that extracellular DNA is modulated and controlled by the two extracellular nucleases Dns and Xds. Our results indicate that extracellular DNA and the extracellular nucleases are involved in diverse processes including the development of a typical biofilm architecture, nutrient acquisition, detachment from biofilms and the colonization fitness of biofilm clumps after ingestion by the host. This study provides new insights into biofilm development and transmission of biofilm-derived V. cholerae. PMID:22032623

DNA Oxidation Profiles of Copper Phenanthrene Chemical Nucleases

NASA Astrophysics Data System (ADS)

Molphy, Zara; Slator, Creina; Chatgilialoglu, Chryssostomos; Kellett, Andrew

2015-04-01

The deleterious effects of metal-catalyzed reactive oxygen species (ROS) in biological systems can be seen in a wide variety of pathological conditions including cancer, cardiovascular disease, ageing, and neurodegenerative disorder. On the other hand however, targeted ROS production in the vicinity of nucleic acids - as demonstrated by metal-activated bleomycin - has paved the way for ROS-active chemotherapeutic drug development. Herein we report mechanistic investigations into the oxidative nuclease activity and redox properties of copper(II) developmental therapeutics [Cu(DPQ)(phen)]2+ (Cu-DPQ-Phen), [Cu(DPPZ)(phen)]2+ (Cu-DPPZ-Phen), and [{Cu(phen)2}2(μ-terph)](terph) (Cu-Terph), with results being compared directly to Sigman’s reagent [Cu(phen)2]2+ throughout (phen = 1,10-phenanthroline; DPQ = dipyridoquinoxaline; DPPZ = dipyridophenazine). Oxidative DNA damage was identified at the minor groove through use of surface bound recognition elements of methyl green, netropsin, and [Co(NH3)6]Cl3 that functioned to control complex accessibility at selected regions. ROS-specific scavengers and stabilisers were employed to identify the cleavage process, the results of which infer hydrogen peroxide produced metal-hydroxo or free hydroxyl radicals (•OH) as the predominant species. The extent of DNA damage owing to these radicals was then quantified through 8-oxo-2'-deoxyguanosine (8-oxo-dG) lesion detection under ELISA protocol with the overall trend following Cu-DPQ-Phen > Cu-Terph > Cu-Phen > Cu-DPPZ. Finally, the effects of oxidative damage on DNA replication processes were investigated using the polymerase chain reaction (PCR) where amplification of 120 base pair DNA sequences of varying base content were inhibited - particularly along A-T rich chains - through oxidative damage of the template strands.

Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma

PubMed Central

Beane, Joal D; Lee, Gary; Zheng, Zhili; Mendel, Matthew; Abate-Daga, Daniel; Bharathan, Mini; Black, Mary; Gandhi, Nimisha; Yu, Zhiya; Chandran, Smita; Giedlin, Martin; Ando, Dale; Miller, Jeff; Paschon, David; Guschin, Dmitry; Rebar, Edward J; Reik, Andreas; Holmes, Michael C; Gregory, Philip D; Restifo, Nicholas P; Rosenberg, Steven A; Morgan, Richard A; Feldman, Steven A

2015-01-01

Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9–84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500–2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted. PMID:25939491

NAD(P)H:quinone oxidoreductase 1 (NQO1) competes with 20S proteasome for binding with C/EBPα leading to its stabilization and protection against radiation-induced myeloproliferative disease.

PubMed

Xu, Junkang; Jaiswal, Anil K

2012-12-07

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein that protects cells against radiation and chemical-induced oxidative stress. Disruption of NQO1 gene in mice leads to increased susceptibility to myeloproliferative disease. In this report, we demonstrate that NQO1 controls the stability of myeloid differentiation factor C/EBPα against 20S proteasomal degradation during radiation exposure stress. Co-immunoprecipitation studies showed that NQO1, C/EBPα, and 20S all interacted with each other. C/EBPα interaction with 20S led to the degradation of C/EBPα. NQO1 in presence of its cofactor NADH protected C/EBPα against 20S degradation. Deletion and site-directed mutagenesis demonstrated that NQO1 and 20S competed for the same binding region (268)SGAGAGKAKKSV(279) in C/EBPα. Mutagenesis studies also revealed that NQO1Y127/Y129 required for NADH binding is essential for NQO1 stabilization of C/EBPα. Exposure of mice and HL-60 cells to 3 Grays of γ-radiation led to increased NQO1 that stabilized C/EBPα against 20S proteasomal degradation. This mechanism of NQO1 regulation of C/EBPα may provide protection to bone marrow against adverse effects of radiation exposure. The studies have significance for human individuals carrying hetero- or homozygous NQO1P187S mutation and are deficient or lack NQO1 protein.

Targeted Mutagenesis of Duplicated Genes in Soybean with Zinc-Finger Nucleases1[W][OA

PubMed Central

Curtin, Shaun J.; Zhang, Feng; Sander, Jeffry D.; Haun, William J.; Starker, Colby; Baltes, Nicholas J.; Reyon, Deepak; Dahlborg, Elizabeth J.; Goodwin, Mathew J.; Coffman, Andrew P.; Dobbs, Drena; Joung, J. Keith; Voytas, Daniel F.; Stupar, Robert M.

2011-01-01

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform—a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting DICER-LIKE (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome. PMID:21464476

Mung bean nuclease: mode of action and specificity vs synthetic esters of 3′-nucleotides

PubMed Central

Kole, R.; Sierakowska, Halina; Szemplińska, Halina; Shugar, D.

1974-01-01

Mung bean nuclease hydrolyzes synthetic esters of 3′-nucleotides to nucleosides and phosphate esters; esters of 2′-nucleotides, and 2′→ 5′ internucleotide linkages, are resistant. Esters of ribonucleotides are cleaved at 100-fold the rate for deoxyribonucleotides, the increased rate being due to presence of the 2′-hydroxyl and not to differences in conformation. Introduction of a 5′-substituent leads to a 3-fold increase in rate. The rates of hydrolysis vary up to 10-fold with the nature of the base, in the order adenine > hypoxanthine > uracil; and up to 6-fold with the nature of the ester radical. This form of cleavage of esters of 3′-nucleotides is also characteristic for nuclease-3′-nucleotidase activities from potato tubers and wheat, suggesting that one type of enzyme is responsible for all these activities. PMID:10793750

The generation of oxidative stress-induced rearrangements in Saccharomyces cerevisiae mtDNA is dependent on the Nuc1 (EndoG/ExoG) nuclease and is enhanced by inactivation of the MRX complex.

PubMed

Dzierzbicki, Piotr; Kaniak-Golik, Aneta; Malc, Ewa; Mieczkowski, Piotr; Ciesla, Zygmunt

2012-12-01

Oxidative stress is known to enhance the frequency of two major types of alterations in the mitochondrial genome of Saccharomyces cerevisiae: point mutations and large deletions resulting in the generation of respiration-deficient petite rhō mutants. We investigated the effect of antimycin A, a well-known agent inducing oxidative stress, on the stability of mtDNA. We show that antimycin enhances exclusively the generation of respiration-deficient petite mutants and this is accompanied by a significant increase in the level of reactive oxygen species (ROS) and in a marked drop of cellular ATP. Whole mitochondrial genome sequencing revealed that mtDNAs of antimycin-induced petite mutants are deleted for most of the wild-type sequence and usually contain one of the active origins of mtDNA replication: ori1, ori2 ori3 or ori5. We show that the frequency of antimycin-induced rhō mutants is significantly elevated in mutants deleted either for the RAD50 or XRS2 gene, both encoding the components of the MRX complex, which is known to be involved in the repair of double strand breaks (DSBs) in DNA. Furthermore, enhanced frequency of rhō mutants in cultures of antimycin-treated cells lacking Rad50 was further increased by the simultaneous absence of the Ogg1 glycosylase, an important enzyme functioning in mtBER. We demonstrate also that rad50Δ and xrs2Δ deletion mutants display a considerable reduction in the frequency of allelic mitochondrial recombination, suggesting that it is the deficiency in homologous recombination which is responsible for enhanced rearrangements of mtDNA in antimycin-treated cells of these mutants. Finally, we show that the generation of large-scale mtDNA deletions induced by antimycin is markedly decreased in a nuc1Δ mutant lacking the activity of the Nuc1 nuclease, an ortholog of the mammalian mitochondrial nucleases EndoG and ExoG. This result indicates that the nuclease plays an important role in processing of oxidative stress

Nuclease digestion and mass spectrometric characterization of oligodeoxyribonucleotides containing 1,2-GpG, 1,2-ApG, and 1,3-GpXpG cisplatin intrastrand cross-links.

PubMed

Williams, Renee T; Nalbandian, Jenifer N; Tu, Audrey; Wang, Yinsheng

2013-05-01

The primary mode of action for cis-diamminedichloroplatinum (II), referred to as cisplatin, toward the treatment of solid malignancies is through formation of cross-links with DNA at purine sites, especially guanines. We prepared oligodeoxyribonucleotides (ODNs) containing a 1,2-GpG, 1,2-ApG, or 1,3-GpXpG cisplatin intrastrand cross-link and the corresponding ODNs modified with (15)N2-labeled cisplatin, and characterized these ODNs with electrospray ionization mass spectrometry (ESI-MS) and tandem MS (MS/MS). We also employed LC-MS/MS to characterize the digestion products of these ODNs after treatment with a cocktail of 4 enzymes (nuclease P1, phosphodiesterases I and II, and alkaline phosphatase). 1,2-GpG was released from the ODNs as a dinucleoside monophosphate or a dinucleotide. Analyses of the digestion products of ODNs containing a 1,2-GpG cross-link on the 5' or 3' terminus revealed that the dinucleotide carries a terminal 5' phosphate. On the other hand, digestion of the 1,3-GpXpG intrastrand cross-link yielded 3 dinucleoside products with 0, 1, or 2 phosphate groups. The availability of the ODNs carrying the stable isotope-labeled lesions, MS/MS analyses of the cisplatin-modified ODNs, and the characterization of the enzymatic digestion products of these ODNs set the stage for the future LC-MS/MS quantification of the 1,2-GpG, 1,2-ApG, and 1,3-GpXpG lesions in cellular DNA. Copyright © 2012 Elsevier B.V. All rights reserved.

Nuclease Digestion and Mass Spectrometric Characterization of Oligodeoxyribonucleotides Containing 1,2-GpG, 1,2-ApG, and 1,3-GpXpG Cisplatin Intrastrand Cross-links

PubMed Central

Williams, Renee T.; Nalbandian, Jenifer; Tu, Audrey; Wang, Yinsheng

2013-01-01

Background The primary mode of action for cis-diamminedichloroplatinum (II), referred to as cisplatin, towards the treatment of solid malignancies is through formation of cross-links with DNA at purine sites, especially guanines. Methods We prepared oligodeoxyribonucleotides (ODNs) containing a 1,2-GpG, 1,2-ApG, or 1,3-GpXpG cisplatin intrastrand cross-link and the corresponding ODNs modified with 15N2-labeled cisplatin, and characterized these ODNs with electrospray ionization mass spectrometry (ESI-MS) and tandem MS (MS/MS). We also employed LC-MS/MS to characterize the digestion products of these ODNs after treatment with a cocktail of 4 enzymes (nuclease P1, phosphodiesterases I and II, and alkaline phosphatase). Results 1,2-GpG was released from the ODNs as a dinucleoside monophosphate or a dinucleotide. Analyses of the digestion products of ODNs containing a 1,2-GpG cross-link on the 5′ or 3′ terminus revealed that the dinucleotide carries a terminal 5′ phosphate. On the other hand, digestion of the 1,3-GpXpG intrastrand cross-link yielded 3 dinucleoside products with 0, 1, or 2 phosphate groups. Results The availability of the ODNs carrying the stable isotope-labeled lesions, MS/MS analyses of the cisplatin-modified ODNs, and the characterization of the enzymatic digestion products of these ODNs set the stage for the future LC-MS/MS quantification of the 1,2-GpG, 1,2-ApG, and 1,3-GpXpG lesions in cellular DNA. PMID:23266768

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

PubMed

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.

Memory Th1 Cells Are Protective in Invasive Staphylococcus aureus Infection

PubMed Central

Lalor, Stephen J.; Leech, John M.; O’Keeffe, Kate M.; Mac Aogáin, Micheál; O’Halloran, Dara P.; Lacey, Keenan A.; Tavakol, Mehri; Hearnden, Claire H.; Fitzgerald-Hughes, Deirdre; Humphreys, Hilary; Fennell, Jérôme P.; van Wamel, Willem J.; Foster, Timothy J.; Geoghegan, Joan A.; Lavelle, Ed C.; Rogers, Thomas R.; McLoughlin, Rachel M.

2015-01-01

Mechanisms of protective immunity to Staphylococcus aureus infection in humans remain elusive. While the importance of cellular immunity has been shown in mice, T cell responses in humans have not been characterised. Using a murine model of recurrent S. aureus peritonitis, we demonstrated that prior exposure to S. aureus enhanced IFNγ responses upon subsequent infection, while adoptive transfer of S. aureus antigen-specific Th1 cells was protective in naïve mice. Translating these findings, we found that S. aureus antigen-specific Th1 cells were also significantly expanded during human S. aureus bloodstream infection (BSI). These Th1 cells were CD45RO+, indicative of a memory phenotype. Thus, exposure to S. aureus induces memory Th1 cells in mice and humans, identifying Th1 cells as potential S. aureus vaccine targets. Consequently, we developed a model vaccine comprising staphylococcal clumping factor A, which we demonstrate to be an effective human T cell antigen, combined with the Th1-driving adjuvant CpG. This novel Th1-inducing vaccine conferred significant protection during S. aureus infection in mice. This study notably advances our understanding of S. aureus cellular immunity, and demonstrates for the first time that a correlate of S. aureus protective immunity identified in mice may be relevant in humans. PMID:26539822

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

PubMed Central

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

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 (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. PMID:24803674

Surveyor nuclease detection of mutations and polymorphisms of mtDNA in children.

PubMed

Pilch, Jacek; Asman, Marek; Jamroz, Ewa; Kajor, Maciej; Kotrys-Puchalska, Elżbieta; Goss, Małgorzata; Krzak, Maria; Witecka, Joanna; Gmiński, Jan; Sieroń, Aleksander L

2010-11-01

Mitochondrial encephalomyopathies are complex disorders with wide range of clinical manifestations. Particularly time-consuming is the identification of mutations in mitochondrial DNA. A group of 20 children with clinical manifestations of mitochondrial encephalomyopathies was selected for molecular studies. The aims were (a) to identify mutations in mtDNA isolated from muscle and (b) to verify detected mutations in DNA isolated from blood, in order to assess the utility of a Surveyor nuclease assay kit for patient screening. The most common changes found were polymorphisms, including a few missense mutations altering the amino acid sequence of mitochondrial proteins. In two boys with MELAS (i.e., mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), a mutation A→G3243 was detected in the tRNALeu gene of mtDNA isolated from muscle and blood. In one boy, the carrier status of his mother was confirmed, based on molecular analysis of DNA isolated from blood. A method using Surveyor nuclease allows systematic screening for small mutations in mtDNA, using as its source blood of the patients and asymptomatic carriers. The method still requires confirmation studying a larger group. In some patients, the use of this method should precede and might limit indications for traumatic muscle and skin biopsy. Copyright © 2010 Elsevier Inc. All rights reserved.

Synthesis of 2',4'-propylene-bridged (carba-ENA) thymidine and its analogues: the engineering of electrostatic and steric effects at the bottom of the minor groove for nuclease and thermodynamic stabilities and elicitation of RNase H.

PubMed

Liu, Yi; Xu, Jianfeng; Karimiahmadabadi, Mansoureh; Zhou, Chuanzheng; Chattopadhyaya, Jyoti

2010-11-05

2',4'-Propylene-bridged thymidine (carba-ENA-T) and five 8'-Me/NH(2)/OH modified carba-ENA-T analogues have been prepared through intramolecular radical addition to C═N of the tethered oxime-ether. These carba-ENA nucleosides have been subsequently incorporated into 15mer oligodeoxynucleotides (AON), and their affinity toward cDNA and RNA, nuclease resistance, and RNase H recruitment capability have been investigated in comparison with those of the native and ENA counterparts. These carba-ENAs modified AONs are highly RNA-selective since all of them led to slight thermal stabilization effect for the AON:RNA duplex, but quite large destabilization effect for the AON:DNA duplex. It was found that different C8' substituents (at the bottom of the minor groove) on carba-ENA-T only led to rather small variation of thermal stability of the AON:RNA duplexes. We, however, observed that the parent carba-ENA-T modified AONs exhibited higher nucleolytic stability than those of the ENA-T modified counterparts. The nucleolytic stability of carba-ENA-T modified AONs can be further modulated by C8' substituent to variable extents depending on not only the chemical nature but also the stereochemical orientation of the C8' substituents: Thus, (1) 8'S-Me on carba-ENA increases the nucleolytic stability but 8'R-Me leads to a decreased effect; (2) 8'R-OH on carba-ENA had little, if any, effect on nuclease resistance but 8'S-OH resulted in significantly decreased nucleolytic stability; and (3) 8'-NH(2) substituted carba-ENA leads to obvious loss in the nuclease resistance. The RNA strand in all of the carba-ENA derivatives modified AON:RNA hybrid duplexes can be digested by RNase H1 with high efficiency, even at twice the rate of those of the native and ENA modified counterpart.

S100A11 protects against neuronal cell apoptosis induced by cerebral ischemia via inhibiting the nuclear translocation of annexin A1.

PubMed

Xia, Qian; Li, Xing; Zhou, Huijuan; Zheng, Lu; Shi, Jing

2018-05-29

The subcellular location of annexin A1 (ANXA1) determines the ultimate fate of neurons after ischemic stroke. ANXA1 nuclear translocation is involved in neuronal apoptosis after cerebral ischemia, and extracellular ANXA1 is also associated with regulation of inflammatory responses. As the factors and mechanism that influence ANXA1 subcellular translocation remain unclear, studies aiming to determine and clarify the role of ANXA1 as a cell fate 'regulator' within cells are critically needed. In this study, we found that intracerebroventricular injection of the recombinant adenovirus vector Ad-S100A11 (carrying S100A11) strongly improved cognitive function and induced robust neuroprotective effects after ischemic stroke in vivo. Furthermore, upregulation of S100A11 protected against neuronal apoptosis induced by oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro. Surprisingly, S100A11 overexpression markedly decreased ANXA1 nuclear translocation and subsequently alleviated OGD/R-induced neuronal apoptosis. Notably, S100A11 exerted its neuroprotective effect by directly binding ANXA1. Importantly, S100A11 directly interacted with ANXA1 through the nuclear translocation signal (NTS) of ANXA1, which is essential for ANXA1 to import into the nucleus. Consistent with our previous studies, ANXA1 nuclear translocation after OGD/R promoted p53 transcriptional activity, induced mRNA expression of the pro-apoptotic Bid gene, and activated the caspase-3 apoptotic pathway, which was almost completely reversed by S100A11 overexpression. Thus, S100A11 protects against cell apoptosis by inhibiting OGD/R-induced ANXA1 nuclear translocation. This study provides a novel mechanism whereby S100A11 protects against neuronal cells apoptosis, suggesting the potential for a previously unidentified treatment strategy in minimizing apoptosis after ischemic stroke.

40 CFR Table 1 to Subpart S of... - General Provisions Applicability to Subpart S a

Code of Federal Regulations, 2010 CFR

2010-07-01

... Subpart S a 1 Table 1 to Subpart S of Part 63 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY..., Subpt. S, Table 1 Table 1 to Subpart S of Part 63—General Provisions Applicability to Subpart S a Reference Applies to Subpart S Comment 63.1(a)(1)-(3) Yes 63.1(a)(4) Yes Subpart S (this table) specifies...

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

PubMed

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.

Functional identification of the non-specific nuclease from white spot syndrome virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Li; Lin Shumei; Yanga Feng

2005-07-05

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 predictedmore » 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.« less

Protection of chickens against infectious bronchitis by a recombinant fowlpox virus co-expressing IBV-S1 and chicken IFNgamma.

PubMed

Wang, Yun-Feng; Sun, Yong-Ke; Tian, Zhan-Cheng; Shi, Xing-Ming; Tong, Guang-Zhi; Liu, Sheng-Wang; Zhi, Hai-Dong; Kong, Xian-Gang; Wang, Mei

2009-11-23

A fowlpox virus expressing the chicken infectious bronchitis virus (IBV) S1 gene of the LX4 strain (rFPV-IBVS1) and a fowlpox virus co-expressing the S1 gene and the chicken type II interferon gene (rFPV-IBVS1-ChIFNgamma) were constructed. These viruses were assessed for their immunological efficacy on specific-pathogen-free (SPF) chickens challenged with a virulent IBV. Although the antibody levels in the rFPV-IBVS1-ChIFNgamma-vaccinated group were lower than those in the attenuated live IB vaccine H120 group and the rFPV-IBVS1 group, the rFPV-IBVS1-ChIFNgamma provided the strongest protection against an IBV LX4 virus challenge (15 out of 16 chickens immunized with rFPV-IBVS1-ChIFNgamma were protected), followed by the attenuated live IB vaccine (13/16 protected) and the rFPV-IBVS1 (12/16 protected). Compared to those of the rFPV-IBVS1 and the attenuated live IB vaccine groups, chickens in the rFPV-IBVS1-ChIFNgamma group eliminated virus more quickly and decreased the presence of viral antigen more significantly in renal tissue. Examination of affected tissues revealed abnormalities in the liver, spleen, kidney, lung and trachea of chickens vaccinated with the attenuated live IB vaccine and the rFPV-IBVS1 vaccine. In rFPV-IBVS1-ChIFNgamma-vaccinated chickens, pathological changes were also observed in those organs, but were milder and lasted shorter. The lesions in the mock control group were the most severe and lasted for at least 20 days. This study demonstrated that chicken type II interferon increased the immunoprotective efficacy of rFPV-IBVS1-ChIFNgamma and normal weight gain in vaccinated chickens although it inhibited serum antibody production.

Peroxynitrite induced formation of the neurotoxins 5-S-cysteinyl-dopamine and DHBT-1: implications for Parkinson's disease and protection by polyphenols.

PubMed

Vauzour, David; Ravaioli, Giulia; Vafeiadou, Katerina; Rodriguez-Mateos, Ana; Angeloni, Cristina; Spencer, Jeremy P E

2008-08-15

Mechanisms of nigral cell injury in Parkinson's disease remain unclear, although a combination of increased oxidative stress, the formation of catecholamine-quinones and the subsequent formation of neurotoxic cysteinyl-catecholamine conjugates may contribute. In the present study, peroxynitrite was observed to generate both 2-S- and 5-S-cysteinyl-dopamine and a dihydrobenzothiazine species, DHBT-1, following the reaction of dopamine with l-cysteine. The formation of 5-S-cysteinyl-dopamine and DHBT-1 in the presence of peroxynitrite induced significant neuronal injury. Pre-treatment of cortical neurons with pelargonidin, quercetin, hesperetin, caffeic acid, the 4'-O-Me derivatives of catechin and epicatechin (0.1-3.0 microM) resulted in concentration dependant protection against 5-S-cysteinyl-dopamine-induced neurotoxicity. These data suggest that polyphenols may protect against neuronal injury induced by endogenous neurotoxins relevant to the aetiology of the Parkinson disease.

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

PubMed Central

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

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.

PubMed

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

Protection of scaffold protein Isu from degradation by the Lon protease Pim1 as a component of Fe-S cluster biogenesis regulation.

PubMed

Ciesielski, Szymon J; Schilke, Brenda; Marszalek, Jaroslaw; Craig, Elizabeth A

2016-04-01

Iron-sulfur (Fe-S) clusters, essential protein cofactors, are assembled on the mitochondrial scaffold protein Isu and then transferred to recipient proteins via a multistep process in which Isu interacts sequentially with multiple protein factors. This pathway is in part regulated posttranslationally by modulation of the degradation of Isu, whose abundance increases >10-fold upon perturbation of the biogenesis process. We tested a model in which direct interaction with protein partners protects Isu from degradation by the mitochondrial Lon-type protease. Using purified components, we demonstrated that Isu is indeed a substrate of the Lon-type protease and that it is protected from degradation by Nfs1, the sulfur donor for Fe-S cluster assembly, as well as by Jac1, the J-protein Hsp70 cochaperone that functions in cluster transfer from Isu. Nfs1 and Jac1 variants known to be defective in interaction with Isu were also defective in protecting Isu from degradation. Furthermore, overproduction of Jac1 protected Isu from degradation in vivo, as did Nfs1. Taken together, our results lead to a model of dynamic interplay between a protease and protein factors throughout the Fe-S cluster assembly and transfer process, leading to up-regulation of Isu levels under conditions when Fe-S cluster biogenesis does not meet cellular demands. © 2016 Ciesielski et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

22 CFR 127.4 - Authority of U.S. Immigration and Customs Enforcement and U.S. Customs and Border Protection...

Code of Federal Regulations, 2010 CFR

2010-04-01

... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Authority of U.S. Immigration and Customs.... Immigration and Customs Enforcement and U.S. Customs and Border Protection officers. (a) U.S. Immigration and.... (b) U.S. Immigration and Customs Enforcement and U.S. Customs and Border Protection officers have the...

Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases

PubMed Central

Merlin, Christine; Beaver, Lauren E.; Taylor, Orley R.; Wolfe, Scot A.; Reppert, Steven M.

2013-01-01

The development of reverse-genetic tools in “nonmodel” insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into “one nucleus” stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and “nonmodel” insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes. PMID:23009861

36 CFR 73.13 - Protection of U.S. World Heritage properties.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 36 Parks, Forests, and Public Property 1 2011-07-01 2011-07-01 false Protection of U.S. World..., DEPARTMENT OF THE INTERIOR WORLD HERITAGE CONVENTION § 73.13 Protection of U.S. World Heritage properties. (a... nominated to the World Heritage List unless its owner concurs in writing to such nomination. The nomination...

Proximal tubule sphingosine kinase-1 has a critical role in A1 adenosine receptor-mediated renal protection from ischemia

PubMed Central

Park, Sang Won; Kim, Mihwa; Kim, Joo Yun; Brown, Kevin M.; Haase, Volker H.; D’Agati, Vivette D.; Lee, H. Thomas

2012-01-01

Renal ischemia reperfusion injury is a major cause of acute kidney injury. We previously found that renal A1 adenosine receptor (A1AR) activation attenuated multiple cell death pathways including necrosis, apoptosis and inflammation. Here, we tested whether induction of cytoprotective sphingosine kinase (SK)-1 and sphingosine-1 phosphate (S1P) synthesis might be the mechanism of protection. A selective A1AR agonist (CCPA) increased the synthesis of S1P and selectively induced SK-1 in mouse kidney and HK-2 cells. This agonist failed to protect SK1-knockout but protected SK2-knockout mice against renal ischemia reperfusion injury indicating a critical role of SK1 in A1AR-mediated renal protection. Inhibition of SK prevented A1AR-mediated defense against necrosis and apoptosis in HK-2 cells. A selective S1P1R antagonist (W146) and global in vivo gene knockdown of S1P1Rs with small interfering RNA completely abolished the renal protection provided by CCPA. Mice selectively deficient in renal proximal tubule S1P1Rs (S1P1Rflox/flox PEPCKCre/−) were not protected against renal ischemia reperfusion injury by CCPA. Mechanistically, CCPA increased nuclear translocation of hypoxia inducible factor-1α in HK-2 cells and selective hypoxia inducible factor-1α inhibition blocked A1AR-mediated induction of SK1. Thus, proximal tubule SK-1 has a critical role in A1AR-mediated protection against renal ischemia reperfusion injury. PMID:22695326

22 CFR 127.4 - Authority of U.S. Immigration and Customs Enforcement and U.S. Customs and Border Protection...

Code of Federal Regulations, 2014 CFR

2014-04-01

... 22 Foreign Relations 1 2014-04-01 2014-04-01 false Authority of U.S. Immigration and Customs Enforcement and U.S. Customs and Border Protection officers. 127.4 Section 127.4 Foreign Relations DEPARTMENT.... Immigration and Customs Enforcement and U.S. Customs and Border Protection officers. (a) U.S. Immigration and...

22 CFR 127.4 - Authority of U.S. Immigration and Customs Enforcement and U.S. Customs and Border Protection...

Code of Federal Regulations, 2012 CFR

2012-04-01

... 22 Foreign Relations 1 2012-04-01 2012-04-01 false Authority of U.S. Immigration and Customs Enforcement and U.S. Customs and Border Protection officers. 127.4 Section 127.4 Foreign Relations DEPARTMENT.... Immigration and Customs Enforcement and U.S. Customs and Border Protection officers. (a) U.S. Immigration and...

22 CFR 127.4 - Authority of U.S. Immigration and Customs Enforcement and U.S. Customs and Border Protection...

Code of Federal Regulations, 2013 CFR

2013-04-01

... 22 Foreign Relations 1 2013-04-01 2013-04-01 false Authority of U.S. Immigration and Customs Enforcement and U.S. Customs and Border Protection officers. 127.4 Section 127.4 Foreign Relations DEPARTMENT.... Immigration and Customs Enforcement and U.S. Customs and Border Protection officers. (a) U.S. Immigration and...

Unifying the DNA End-processing Roles of the Artemis Nuclease

PubMed Central

Chang, Howard H. Y.; Watanabe, Go; Lieber, Michael R.

2015-01-01

Artemis is a member of the metallo-β-lactamase protein family of nucleases. It is essential in vertebrates because, during V(D)J recombination, the RAG complex generates hairpins when it creates the double strand breaks at V, D, and J segments, and Artemis is required to open the hairpins so that they can be joined. Artemis is a diverse endo- and exonuclease, and creating a unified model for its wide range of nuclease properties has been challenging. Here we show that Artemis resects iteratively into blunt DNA ends with an efficiency that reflects the AT-richness of the DNA end. GC-rich ends are not cut by Artemis alone because of a requirement for DNA end breathing (and confirmed using fixed pseudo-Y structures). All DNA ends are cut when both the DNA-dependent protein kinase catalytic subunit and Ku accompany Artemis but not when Ku is omitted. These are the first biochemical data demonstrating a Ku dependence of Artemis action on DNA ends of any configuration. The action of Artemis at blunt DNA ends is slower than at overhangs, consistent with a requirement for a slow DNA end breathing step preceding the cut. The AT sequence dependence, the order of strand cutting, the length of the cuts, and the Ku-dependence of Artemis action at blunt ends can be reconciled with the other nucleolytic properties of both Artemis and Artemis·DNA-PKcs in a model incorporating DNA end breathing of blunt ends to form transient single to double strand boundaries that have structural similarities to hairpins and fixed 5′ and 3′ overhangs. PMID:26276388

Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

PubMed Central

Vallerga, María Belén; Mansilla, Sabrina F.; Federico, María Belén; Bertolin, Agustina P.; Gottifredi, Vanesa

2015-01-01

After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates. PMID:26627254

FTY720 Protects Cardiac Microvessels of Diabetes: A Critical Role of S1P1/3 in Diabetic Heart Disease

PubMed Central

Wei, Liping; Gao, Haokao; Zhang, Rongqing; Tao, Ling; Cao, Feng; Wang, Haichang

2012-01-01

Background: Diabetes is associated with an increased risk of cardiac microvascular disease. The mechanisms by which this damage occurs are unknown. However, research suggests that signaling through the sphingosine-1-phosphates receptor 1 and 3 (S1P1/3) by FTY720, a sphiongolipid drug that is structually similar to SIP, may play a role in the treatment on cardiac microvascular dysfunction in diabetes. We hypothesized that FTY720 might exert the cardioprotective effects of S1P1 and S1P3 viaprotein kinase C-beta (PKCβ II) signaling pathway. Methodology/Principal Findings: Transthoracic echocardiography was performed to detect the change of cardiac function. Scanning and transmission electron microscope with lanthanum tracer were used to determine microvascular ultrastructure and permeability in vivo. Apoptosis was detected by TUNEL and CD31 dual labeling in paraffin-embedded sections. Laser capture miscrodissection was used to assess cardiac micovascular endothelial cells (CMECs) in vivo. RT-PCR and Western blot analysis were used to determine the mRNA levels and protein expression of S1P1, S1P3, and PKCβ II. In the diabetic rats vs. controls, cardiac capillaries showed significantly higher density; CD31 positive endothelial cells were significantly reduced; the apoptosis index of cardiac endothlial cells was significantly higher. And FTY720 could increase the expressional level of S1P1 and boost S1P3 trasnslocation from membrane to nuclear, then ameliorate cardiac microvascular barrier impairment and pathologic angiogenesis induced by diabetes. In addition, overexpression of PKCβ II significantly decreased the protective effect of FTY720. Conclusions: Our study represents that the deregulation of S1P1 and S1P3 is an important signalresponsible for cardiac microvascular dysfunction in diabetes. FTY720 might be competent to serve as a potential therapeutic approach for diabetic heart disease through ameliorating cardiac microvascular barrier impairment and

Myostatin gene mutated mice induced with tale nucleases.

PubMed

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

2015-01-01

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

76 FR 65207 - U.S. Customs and Border Protection

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-20

... DEPARTMENT OF HOMELAND SECURITY U.S. Customs and Border Protection Notice of Cancellation of Customs Broker Licenses AGENCY: U.S. Customs and Border Protection, U.S. Department of Homeland Security.... 1641) and the U.S. Customs and Border Protection regulations (19 CFR 111.51), the following Customs...

BacMam virus-based surface display of the infectious bronchitis virus (IBV) S1 glycoprotein confers strong protection against virulent IBV challenge in chickens.

PubMed

Zhang, Jie; Chen, Xiao-Wei; Tong, Tie-Zhu; Ye, Yu; Liao, Ming; Fan, Hui-Ying

2014-02-03

Avian infectious bronchitis virus (IBV) is associated with production inefficiencies in domestic fowl, and causes massive economic losses to the poultry industry worldwide. Progress has been made in designing novel and efficient candidate vaccines to control IBV infection. BacMam virus, a modified baculovirus mediating transgene expression under the control of a mammalian promoter, has emerged as a versatile and safe vector during vaccine development. In previous work, we generated the BacMam virus Ac-CMV-S1, which expressed the S1 glycoprotein of IBV-M41. We showed that Ac-CMV-S1 induced excellent cellular immunity, but did not confer adequate protection in chickens compared with the conventional inactivated vaccine. In the current study, we generated an improved BacMam virus, BV-Dual-S1. This virus displayed the S1 glycoprotein on the baculovirus envelope, and was capable of expressing it in mammalian cells. BV-Dual-S1 elicited stronger humoral and cell-mediated immune responses, and showed greater capacity for induction of cytotoxic T lymphocyte responses, compared with Ac-CMV-S1 in specific pathogen-free chickens. A significant difference was not observed for protection rates between chickens immunized with BV-Dual-S1 (83%) or inactivated vaccine (89%) following challenge with virulent IBV-M41. Our findings show that the protective efficacy of BV-Dual-S1 could be significantly enhanced by baculovirus display technology. BacMam virus-based surface display strategies could serve as effective tools in designing vaccines against IB and other infectious diseases. Copyright © 2013. Published by Elsevier Ltd.

MiR-361-5p inhibits colorectal and gastric cancer growth and metastasis by targeting staphylococcal nuclease domain containing-1

PubMed Central

Zhang, Yuxin; Zheng, Yasheng; Lin, Chengchun; Wu, Ying; Guan, Guijie; Sha, Ruihua; Zhou, Qingxin; Wang, Dejun; Zhou, Xinglu; Li, Juan; Qiu, Xiaohui

2015-01-01

MicroRNAs (miRs) function as key regulators of gene expression and their deregulation is associated with the carcinogenesis of various cancers. In the present study, we investigated the biological role and mechanism of miR-361-5p in colorectal carcinoma (CRC) and gastric cancer (GC). We showed that microRNA-361-5p (miR-361-5p) was down-regulated in CRC and GC in comparison to the controls. Meanwhile, the expression levels of miR-361-5p negatively correlated with lung metastasis and prognosis in clinical CRC patients. Overexpression of miR-361-5p markedly suppressed proliferation, migration and invasion of cancer cells. Additionally, this phenotype could be partially rescued by the ectopic expression of staphylococcal nuclease domain containing-1 (SND1). SND1 was identified as a target of miR-361-5p using bioinformatics analysis and in vitro luciferase reporter assays. In turn, SND1 bound to pre-miR-361-5p and suppressed the expression of miR-361-5p, thus exerting a feedback loop. Most interestingly, in vivo studies showed that restoration of miR-361-5p significantly inhibited tumor growth and especially the lung metastasis in nude mice. Therefore, it could be concluded that miR-361-5p functions as a tumor-suppressive miRNA through directly binding to SND1, highlighting its potential as a novel agent for the treatment of patients with CRC and GC. PMID:25965817

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

PubMed Central

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

78 FR 35044 - U.S. Customs and Border Protection

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-11

... DEPARTMENT OF HOMELAND SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Visa Waiver Program Carrier Agreement (CBP Form I-775) AGENCY: U.S. Customs and Border Protection... information collection: 1651-0110. SUMMARY: U.S. Customs and Border Protection (CBP) of the Department of...

Sulforaphane protects cortical neurons against 5-S-cysteinyl-dopamine-induced toxicity through the activation of ERK1/2, Nrf-2 and the upregulation of detoxification enzymes.

PubMed

Vauzour, David; Buonfiglio, Maria; Corona, Giulia; Chirafisi, Joselita; Vafeiadou, Katerina; Angeloni, Cristina; Hrelia, Silvana; Hrelia, Patrizia; Spencer, Jeremy P E

2010-04-01

The degeneration of dopaminergic neurons in the substantia nigra has been linked to the formation of the endogenous neurotoxin 5-S-cysteinyl-dopamine. Sulforaphane (SFN), an isothiocyanate derived from the corresponding precursor glucosinolate found in cruciferous vegetables has been observed to exert a range of biological activities in various cell populations. In this study, we show that SFN protects primary cortical neurons against 5-S-cysteinyl-dopamine induced neuronal injury. Pre-treatment of cortical neurons with SFN (0.01-1 microM) resulted in protection against 5-S-cysteinyl-dopamine-induced neurotoxicity, which peaked at 100 nM. This protection was observed to be mediated by the ability of SFN to modulate the extracellular signal-regulated kinase 1 and 2 and the activation of Kelch-like ECH-associated protein 1/NF-E2-related factor-2 leading to the increased expression and activity of glutathione-S-transferase (M1, M3 and M5), glutathione reductase, thioredoxin reductase and NAD(P)H oxidoreductase 1. These data suggest that SFN stimulates the NF-E2-related factor-2 pathway of antioxidant gene expression in neurons and may protect against neuronal injury relevant to the aetiology of Parkinson's disease.

Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

PubMed Central

Ochiai, Hiroshi; Sakamoto, Naoaki; Fujita, Kazumasa; Nishikawa, Masatoshi; Suzuki, Ken-ichi; Matsuura, Shinya; Miyamoto, Tatsuo; Sakuma, Tetsushi; Shibata, Tatsuo; Yamamoto, Takashi

2012-01-01

To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B–GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos. PMID:22711830

Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation.

PubMed

Musharova, Olga; Klimuk, Evgeny; Datsenko, Kirill A; Metlitskaya, Anastasia; Logacheva, Maria; Semenova, Ekaterina; Severinov, Konstantin; Savitskaya, Ekaterina

2017-04-07

During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR interference machinery, which in the case of Type I CRISPR-Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1-Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Trotter welfare’s protection: A legislative perspective

PubMed Central

Passantino, Annamaria; Giannetto, Claudia; Passantino, Letizia; Piccione, Giuseppe

2015-01-01

The Council of Europe’s activities in the field of animal welfare are particularly noteworthy and comprise the elaboration of several norms for the protection of animals. Concerning the specific European Directive, Regulations or Convention for the protection of animals, the Authors underline the missing of specifics recommendations concerning the welfare of sport horses and especially of trotters. Guidelines are reported by regulation of equestrian sports. The paper’s purpose is to give practical elements to individuate the welfare state and to promote a clear regulation on welfare, care and protection of trotters. PMID:27047109

The nuclease hSNM1B/Apollo is linked to the Fanconi anemia pathway via its interaction with FANCP/SLX4.

PubMed

Salewsky, Bastian; Schmiester, Maren; Schindler, Detlev; Digweed, Martin; Demuth, Ilja

2012-11-15

The recessive genetic disorder Fanconi anemia (FA) is clinically characterized by congenital defects, bone marrow failure and an increased incidence of cancer. Cells derived from FA patients exhibit hypersensitivity to DNA interstrand crosslink (ICL)-inducing agents. We have earlier reported a similar cellular phenotype for human cells depleted of hSNM1B/Apollo (siRNA). In fact, hSNM1B/Apollo has a dual role in the DNA damage response and in generation and maintenance of telomeres, the latter function involving interaction with the shelterin protein TRF2. Here we find that ectopically expressed hSNM1B/Apollo co-immunoprecipitates with SLX4, a protein recently identified as a new FA protein, FANCP, and known to interact with several structure-specific nucleases. As shown by immunofluorescence analysis, FANCP/SLX4 depletion (siRNA) resulted in a significant reduction of hSNM1B/Apollo nuclear foci, supporting the functional relevance of this new protein interaction. Interestingly, as an additional consequence of FANCP/SLX4 depletion, we found a reduction of cellular TRF2, in line with its telomere-related function. Finally, analysis of human cells following double knockdown of hSNM1B/Apollo and FANCP/SLX4 indicated that they function epistatically. These findings further substantiate the role of hSNM1B/Apollo in a downstream step of the FA pathway during the repair of DNA ICLs.

TRF2 Recruits RTEL1 to Telomeres in S Phase to Promote T-Loop Unwinding

PubMed Central

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 TRF2-RTEL1 interaction is mediated by a metal-coordinating C4C4 motif in RTEL1, which is compromised by the Hoyeraal-Hreidarsson syndrome (HHS) mutation, RTEL1R1264H. Conversely, we define a TRF2I124D substitution mutation within the TRFH domain of TRF2, which eliminates RTEL1 binding and phenocopies the RTEL1R1264H mutation, giving rise to aberrant t-loop excision, telomere length heterogeneity, and loss of the telomere as a circle. These results implicate TRF2 in the recruitment of RTEL1 to facilitate t-loop disassembly at telomeres in S phase. PMID:25620558

TRF2 recruits RTEL1 to telomeres in S phase to promote t-loop unwinding.

PubMed

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

2015-02-19

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 TRF2-RTEL1 interaction is mediated by a metal-coordinating C4C4 motif in RTEL1, which is compromised by the Hoyeraal-Hreidarsson syndrome (HHS) mutation, RTEL1(R1264H). Conversely, we define a TRF2(I124D) substitution mutation within the TRFH domain of TRF2, which eliminates RTEL1 binding and phenocopies the RTEL1(R1264H) mutation, giving rise to aberrant t-loop excision, telomere length heterogeneity, and loss of the telomere as a circle. These results implicate TRF2 in the recruitment of RTEL1 to facilitate t-loop disassembly at telomeres in S phase. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

SPECIES-SPECIFIC DETECTION OF THREE HUMAN-PATHOGENIC MICROSPORIDIAL SPECIES FROM THE GENUS ENCEPHALITOZOON VIA FLUOROGENIC 5' NUCLEASE PCR ASSAYS

EPA Science Inventory

This describes fluorogenic 5' nuclease PCR assays suitable for rapid, sensitive, quantitative, high-throughput detection of the human-pathogenic microsporidial species Encephalitozoon hellem, E. cunicli and E. intestinalis. The assays utilize species-specific primer sets and a g...

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

PubMed

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

Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism

PubMed Central

Zhao, Haiyan; Lin, Zihan; Lynn, Anna Y.; Varnado, Brittany; Beutler, John A.; Murelli, Ryan P.; Le Grice, Stuart F. J.; Tang, Liang

2015-01-01

Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg2+. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg2+ from Ca2+. Using a metal ion chelator β-thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution. PMID:26450964

Hospital respiratory protection practices in 6 U.S. states: A public health evaluation study

PubMed Central

Peterson, Kristina; Novak, Debra; Stradtman, Lindsay; Wilson, David; Couzens, Lance

2015-01-01

Background Lessons learned from the influenza A (H1N1) virus revealed a need to better understand hospitals’ respiratory protection programmatic practice gaps. This article reports findings from a multistate assessment of hospitals’ adherence to the Occupational Safety and Health Administration’s respiratory protection program (RPP) requirements and the Centers for Disease Control and Prevention’s infection control guidance. Methods Onsite surveys were conducted in 98 acute care hospitals in 6 U.S. states, including >1,500 hospital managers, unit managers, and health care workers. Descriptive statistics were used to assess hospital adherence. Results Most acute care hospitals adhere to requirements for initial medical evaluations, fit testing, training, and recommended respiratory protection when in close contact with patients who have suspected or confirmed seasonal influenza. Low hospital adherence was found for respiratory protection with infectious diseases requiring airborne precautions, aerosol-generating procedures with seasonal influenza, and checking of the respirator’s user seal. Hospitals’ adherence was also low with follow-up program evaluations, medical re-evaluations, and respirator maintenance. Conclusion Efforts should be made to closely examine ways of strengthening hospitals’ RPPs to ensure the program’s ongoing effectiveness and workers’ proper selection and use of respiratory protection. Implications for improved RPPs and practice are discussed. PMID:25564126

Characterization of cross protection of Swine-Origin Influenza Virus (S-OIV) H1N1 and reassortant H5N1 influenza vaccine in BALB/c mice given a single-dose vaccination

PubMed Central

2013-01-01

Background Influenza virus has antigen drift and antigen shift effect, vaccination with some influenza vaccine might not induce sufficient immunity for host to the threat of other influenza virus strains. S-OIV H1N1 and H5N1 influenza vaccines in single-dose immunization were evaluated in mice for cross protection to the challenge of A/California/7/2009 H1N1 or NIBRG-14 H5N1 virus. Results Both H1N1 and H5N1 induced significant homologous IgG, HAI, and microneutralization antibody responses in the mice, while only vaccines plus adjuvant produced significant heterogeneous IgG and HAI antibody responses. Both alum and MPLA adjuvants significantly reduced the S-OIV H1N1 vaccine dose required to elicit protective HAI antibody titers from 0.05 μg to 0.001 μg. Vaccines alone did not protect mice from challenge with heterogeneous influenza virus, while H5N1 vaccine plus alum and MPLA adjuvants did. Mouse body weight loss was also less significant in the presence of adjuvant than in the vaccine without adjuvant. Furthermore, both H1N1 and H5N1 lung viral titers of immunized mice were significantly reduced post challenge with homologous viruses. Conclusion Only in the presence of MPLA adjuvant could the H5N1 vaccine significantly reduce mouse lung viral titers post H1N1 virus challenge, and not vice versa. MPLA adjuvant induced cross protection with a single dose vaccination to the challenge of heterogeneous influenza virus in mice. Lung viral titer seemed to be a better indicator compared to IgG, neutralization antibody, and HAI titer to predict survival of mice infected with influenza virus. PMID:23517052

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

PubMed

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.

Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2

PubMed Central

White, Rebekah R.; Shan, Siqing; Rusconi, Christopher P.; Shetty, Geetha; Dewhirst, Mark W.; Kontos, Christopher D.; Sullenger, Bruce A.

2003-01-01

Angiopoietin-2 (Ang2) appears to be a naturally occurring antagonist of the endothelial receptor tyrosine kinase Tie2, an important regulator of vascular stability. Destabilization of the endothelium by Ang2 is believed to potentiate the actions of proangiogenic growth factors. To investigate the specific role of Ang2 in the adult vasculature, we generated a nuclease-resistant RNA aptamer that binds and inhibits Ang2 but not the related Tie2 agonist, angiopoietin-1. Local delivery of this aptamer but not a partially scrambled mutant aptamer inhibited basic fibroblast growth factor-mediated neovascularization in the rat corneal micropocket angiogenesis assay. These in vivo data directly demonstrate that a specific inhibitor of Ang2 can act as an antiangiogenic agent. PMID:12692304

A Nuclease from Streptococcus mutans Facilitates Biofilm Dispersal and Escape from Killing by Neutrophil Extracellular Traps

PubMed Central

Liu, Jia; Sun, Luping; Liu, Wei; Guo, Lihong; Liu, Zhaohui; Wei, Xi; Ling, Junqi

2017-01-01

Streptococcus mutans is the primary etiologic agent of dental caries and occasionally infective endocarditis, with the ability to form biofilms and disperse cells into distal sites to exacerbate and spread infection. In this study, we identified a nuclease (DeoC) as a S. mutans biofilm dispersal modulating factor through microarray analysis. In vitro assays revealed a dispersal defect of a deoC deletion mutant, and functional studies with purified protein were indicative of the biofilm dispersal activity of DeoC. Neutrophils are a key host response factor restraining bacterial spreading through the formation of neutrophil extracellular traps (NETs), which consist of a nuclear DNA backbone associated with antimicrobial peptides. Therefore, we hypothesized that the dispersed S. mutans might utilize DeoC to degrade NETs and escape killing by the immune system. It was found that S. mutans induced NET formation upon contact with neutrophils, while the presence of NETs in turn enhanced the deoC expression of S. mutans. Fluorescence microscopy inspection showed that deoC deletion resulted in a decreased NET degradation ability of S. mutans and enhanced susceptibility to neutrophil killing. Data obtained from this study assigned two important roles for DeoC in S. mutans: contributing to the spread of infection through mediating biofilm dispersal, and facilitating the escape of S. mutans from neutrophil killing through NET degradation. PMID:28401067

A Nuclease from Streptococcus mutans Facilitates Biofilm Dispersal and Escape from Killing by Neutrophil Extracellular Traps.

PubMed

Liu, Jia; Sun, Luping; Liu, Wei; Guo, Lihong; Liu, Zhaohui; Wei, Xi; Ling, Junqi

2017-01-01

Streptococcus mutans is the primary etiologic agent of dental caries and occasionally infective endocarditis, with the ability to form biofilms and disperse cells into distal sites to exacerbate and spread infection. In this study, we identified a nuclease (DeoC) as a S. mutans biofilm dispersal modulating factor through microarray analysis. In vitro assays revealed a dispersal defect of a deoC deletion mutant, and functional studies with purified protein were indicative of the biofilm dispersal activity of DeoC. Neutrophils are a key host response factor restraining bacterial spreading through the formation of neutrophil extracellular traps (NETs), which consist of a nuclear DNA backbone associated with antimicrobial peptides. Therefore, we hypothesized that the dispersed S. mutans might utilize DeoC to degrade NETs and escape killing by the immune system. It was found that S. mutans induced NET formation upon contact with neutrophils, while the presence of NETs in turn enhanced the deoC expression of S. mutans . Fluorescence microscopy inspection showed that deoC deletion resulted in a decreased NET degradation ability of S. mutans and enhanced susceptibility to neutrophil killing. Data obtained from this study assigned two important roles for DeoC in S. mutans : contributing to the spread of infection through mediating biofilm dispersal, and facilitating the escape of S. mutans from neutrophil killing through NET degradation.

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

PubMed Central

Shah, Megha; Mejdani, Marios; Calmettes, Charles; Moraes, Trevor F.

2017-01-01

ABSTRACT 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. PMID:29233895

RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency.

PubMed

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

PubMed Central

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

Planetary protection policy (U.S.A.)

NASA Technical Reports Server (NTRS)

Rummel, John D.

1992-01-01

Through existing treaty obligations of the United States, NASA is committed to exploring space while avoiding biological contamination of the planets, and to the protection of the earth against harm from materials returned from space. Because of the similarities between Mars and earth, plans for the exploration of Mars evoke discussions of these Planetary Protection issues. U.S. Planetary Protection Policy will be focused on the preservation of these goals in an arena that will change with the growth of scientific knowledge about the Martian environment. Early opportunities to gain the appropriate data will be used to guide later policy implementation. Because human presence on Mars will result in the end of earth's separation from the Martian environment, it is expected that precursor robotic missions will address critical planetary protection concerns before humans arrive.

Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay.

PubMed

Li, Chia-Lung; Yang, Wei-Zen; Shi, Zhonghao; Yuan, Hanna S

2018-05-01

Tudor staphylococcal nuclease (TSN) is an evolutionarily conserved ribonuclease in eukaryotes that is composed of five staphylococcal nuclease-like domains (SN1-SN5) and a Tudor domain. TSN degrades hyper-edited double-stranded RNA, including primary miRNA precursors containing multiple I•U and U•I pairs, and mature miRNA during miRNA decay. However, how TSN binds and degrades its RNA substrates remains unclear. Here, we show that the C. elegans TSN (cTSN) is a monomeric Ca 2+ -dependent ribonuclease, cleaving RNA chains at the 5'-side of the phosphodiester linkage to produce degraded fragments with 5'-hydroxyl and 3'-phosphate ends. cTSN degrades single-stranded RNA and double-stranded RNA containing mismatched base pairs, but is not restricted to those containing multiple I•U and U•I pairs. cTSN has at least two catalytic active sites located in the SN1 and SN3 domains, since mutations of the putative Ca 2+ -binding residues in these two domains strongly impaired its ribonuclease activity. We further show by small-angle X-ray scattering that rice osTSN has a flexible two-lobed structure with open to closed conformations, indicating that TSN may change its conformation upon RNA binding. We conclude that TSN is a structure-specific ribonuclease targeting not only single-stranded RNA, but also unstructured regions of double-stranded RNA. This study provides the molecular basis for how TSN cooperates with RNA editing to eliminate duplex RNA in cell defense, and how TSN selects and degrades RNA during microRNA decay. © 2018 Li et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Telomeric 3' overhangs derive from resection by Exo1 and Apollo and fill-in by POT1b-associated CST.

PubMed

Wu, Peng; Takai, Hiroyuki; de Lange, Titia

2012-07-06

A 3' overhang is critical for the protection and maintenance of mammalian telomeres, but its synthesis must be regulated to avoid excessive resection of the 5' end, which could cause telomere shortening. How this balance is achieved in mammals has not been resolved. Here, we determine the mechanism for 3' overhang synthesis in mouse cells by evaluating changes in telomeric overhangs throughout the cell cycle and at leading- and lagging-end telomeres. Apollo, a nuclease bound to the shelterin subunit TRF2, initiates formation of the 3' overhang at leading-, but not lagging-end telomeres. Hyperresection by Apollo is blocked at both ends by the shelterin protein POT1b. Exo1 extensively resects both telomere ends, generating transient long 3' overhangs in S/G2. CST/AAF, a DNA polα.primase accessory factor, binds POT1b and shortens the extended overhangs produced by Exo1, likely through fill-in synthesis. 3' overhang formation is thus a multistep, shelterin-controlled process, ensuring functional telomeric overhangs at chromosome ends. Copyright © 2012 Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Masahito; Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571; Umeyama, Kazuhiro

2010-11-05

Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} 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 themore » 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.« less

Protection against oxidative DNA damage and stress in human prostate by glutathione S-transferase P1

PubMed Central

Kanwal, Rajnee; Pandey, Mitali; Bhaskaran, Natarajan; MacLennan, Gregory T; Fu, Pingfu; Ponsky, Lee E; Gupta, Sanjay

2014-01-01

The pi-class glutathione S-transferase (GSTP1) actively protect cells from carcinogens and electrophilic compounds. Loss of GSTP1 expression via promoter hypermethylation is the most common epigenetic alteration observed in human prostate cancer. Silencing of GSTP1 can increase generation of reactive oxygen species (ROS) and DNA damage in cells. In this study we investigated whether loss of GSTP1 contributes to increased DNA damage that may predispose men to a higher risk of prostate cancer. We found significantly elevated (103%; P<0.0001) levels of 8-oxo-2′-deoxogunosine (8-OHdG), an oxidative DNA damage marker, in adenocarcinomas, compared to benign counterparts, which positively correlated (r=0.2) with loss of GSTP1 activity (34%; P<0.0001). Silencing of GSTP1 using siRNA approach in normal human prostate epithelial RWPE1 cells caused increased intracellular production of ROS and higher susceptibility of cells to H2O2-mediated oxidative stress. Additionally, human prostate carcinoma LNCaP cells, which contain a silenced GSTP1 gene, were genetically modified to constitutively express high levels of GSTP1. Induction of GSTP1 activity lowered endogenous ROS levels in LNCaP-pLPCX-GSTP1 cells, and when exposed to H2O2, these cells exhibited significantly reduced production of ROS and 8-OHdG levels, compared to vector control LNCaP-pLPCX cells. Furthermore, exposure of LNCaP cells to green tea polyphenols caused re-expression of GSTP1, which protected the cells from H2O2-mediated DNA damage through decreased ROS production compared to non-exposed cells. These results suggest that loss of GSTP1 expression in human prostate cells, a process that increases their susceptibility to oxidative stress-induced DNA damage, may be an important target for primary prevention of prostate cancer. PMID:22833520

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

PubMed

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.

Anticonvulsant activity of a mGlu(4alpha) receptor selective agonist, (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid.

PubMed

Chapman, A G; Talebi, A; Yip, P K; Meldrum, B S

2001-07-20

The metabotropic Group III agonist, (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid (ACPT-1), selective for the mGlu(4alpha) receptor, suppresses sound-induced seizures in DBA/2 mice following its intracerebroventricular (i.c.v.) administration (ED(50) 5.6 [2.9-10.7], nmol i.c.v., 15 min, clonic phase) and in genetically epilepsy-prone (GEP) rats following focal administration into the inferior colliculus (ED(50) 0.08 [0.01-0.50], nmol, 60 min, clonic phase). ACPT-1 also protects against clonic seizures induced in DBA/2 mice by the Group I agonist, (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG) (ED(50) 0.60 [0.29-1.2], nmol i.c.v.) and by the Group III antagonist, (RS)-alpha-methylserine-O-phosphate (MSOP) (ED(50) 49.3 [37.9-64.1], nmol i.c.v.). Another Group III agonist, (RS)-4-phosphonophenyl-glycine (PPG), preferentially activating the mGlu(8) receptor, previously shown to protect against sound-induced seizures in DBA/2 mice and GEP rats, also protects against seizures induced in DBA/2 by 3,5-DHPG (ED(50) 3.7 [2.4-5.7], nmol i.c.v.) and by the Group III antagonist, MSOP (ED(50) 40.2 [21.0-77.0], nmol i.c.v.). At very high doses (500 nmol i.c.v. and above), Group III antagonists have pro-convulsant and convulsant activity. The anticonvulsant protection against sound-induced seizures in DBA/2 mice provided by a fully protective dose (20 nmol, i.c.v.) of the mGlu(4) receptor agonist ACPT-1, is partially reversed by the co-administration of the Group III antagonists, MSOP, (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG) or (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4), in the 20-50 nmol dose range. At doses of 50-200 nmol, MPPG and MAP4 cause further reversal of the ACPT-1 anticonvulsant protection, while the MSOP effect on ACPT-1 protection is abolished at higher doses. In contrast, the anticonvulsant protection against sound-induced seizures in DBA/2 mice provided by a fully protective dose (20 nmol, i.c.v.) of the mGlu(8) receptor agonist PPG, is not

Telomeric 3′ overhangs derive from resection by Exo1 and Apollo and fill-in by POT1b-associated CST

PubMed Central

Wu, Peng; Takai, Hiroyuki; de Lange, Titia

2012-01-01

SUMMARY A 3′ overhang is critical for the protection and maintenance of mammalian telomeres. How these overhangs are generated and whether different processing steps modify telomeres synthesized by leading- and lagging-strand DNA replication was not known. Here we evaluate changes in the telomeric overhangs through the cell cycle and at leading- and lagging-end telomeres in mouse cells lacking relevant genes. Apollo, a nuclease bound to the shelterin subunit TRF2, initiated formation of the 3′ overhang at leading-, but not lagging-end telomeres. Hyper-resection by Apollo was blocked at both ends by the shelterin protein POT1b. Exo1 extensively resected both telomere ends, generating long 3′ overhangs that transiently occurred in S/G2. CST/AAF, a DNA polymeraseα. primase accessory factor related to yeast CST, bound POT1b and shortened the extended overhangs produced by Exo1, most likely through fill-in synthesis. The results establish 3′ overhang formation as a multi-step, shelterin-controlled process that ensures functional telomeric overhangs at all chromosome ends. PMID:22748632

Modeling of the Human Alveolar Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nuclease

PubMed Central

Lagutina, Irina V.; Valentine, Virginia; Picchione, Fabrizio; Harwood, Frank; Valentine, Marcus B.; Villarejo-Balcells, Barbara; Carvajal, Jaime J.; Grosveld, Gerard C.

2015-01-01

Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice. PMID:25659124

FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5′-DNA end

PubMed Central

Sato, Koichi; Shimomuki, Mayo; Katsuki, Yoko; Takahashi, Daisuke; Kobayashi, Wataru; Ishiai, Masamichi; Miyoshi, Hiroyuki; Takata, Minoru; Kurumizaka, Hitoshi

2016-01-01

The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork. PMID:27694619

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, T; Huang, S; Zhao, XF

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

S-protected thiolated hydroxyethyl cellulose (HEC): Novel mucoadhesive excipient with improved stability.

PubMed

Leonaviciute, Gintare; Bonengel, Sonja; Mahmood, Arshad; Ahmad Idrees, Muneeb; Bernkop-Schnürch, Andreas

2016-06-25

The aim of this study was the design of novel S-protected thiolated hydroxyethyl cellulose (HEC) and the assessment of its mucoadhesive properties and biodegradability compared to the corresponding unmodified polymer. Thiolated HEC was S-protected via disulfide bond formation between 6-mercaptonicotinamide (6-MNA) and the thiol substructures of the polymer. In vitro screening of mucoadhesive properties was accomplished using two different methods: rotating cylinder studies and viscosity measurements. Moreover, biodegradability of these polymers by cellulase, xylanase and lysozyme was evaluated. MTT and LDH assays were performed on Caco-2 cells to determine the cytotoxicity of S-protected thiolated HEC. Thiolated HEC displayed 280.09±1.70μmol of free thiol groups per gram polymer. S-protected thiolated HEC exhibiting 270.8±21.11μmol immobilized 6-MNA ligands per gram of polymer was shown being 2.4-fold more mucoadhesive compared to thiolated HEC. No mucoadhesion was observed in case of unmodified HEC. Results were in a good agreement with rheological studies. The presence of free thiol moieties likely caused lower degree of hydrolysis by xylanase, whereas the degradation by both enzymes cellulase and xylanase was more hampered when 6-MNA was introduced as ligand for thiol group's protection. Findings in cell viability revealed that all three conjugates were non-toxic. S-protection of thiolated hydroxyethyl cellulose improved mucoadhesive properties and provided pronounced stability towards enzymatic attack, that makes this excipient superior for non-invasive drug administration over thiolated and unmodified forms. Copyright © 2016 Elsevier Ltd. All rights reserved.

The E. coli 16S rRNA binding site of ribosomal protein S15: higher-order structure in the absence and in the presence of the protein.

PubMed Central

Mougel, M; Philippe, C; Ebel, J P; Ehresmann, B; Ehresmann, C

1988-01-01

We have investigated in detail the secondary and tertiary structures of E. coli 16S rRNA binding site of protein S15 using a variety of enzymatic and chemical probes. RNase T1 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with 1-cyclohexyl-3 (2-(1-methylmorpholino)-ethyl)-carboiimide-p- toluenesulfonate (at U(N-3) and G(N-1)) and with diethylpyrocarbonate (at A(N-7)). The RNA region corresponding to nucleotides 652 to 753 was tested within: (1) the complete 16S rRNA molecule; (2) a 16S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S15-16S rRNA complex; (4) the S15-fragment complex. Cleavage and modification sites were detected by primer extension with reverse transcriptase. Our results show that: (1) The synthetized fragment folds into the same overall secondary structure as in the complete 16S rRNA, with the exception of the large asymmetrical internal loop (nucleotides 673-676/714-733) which is fully accessible in the fragment while it appears conformationally heterogeneous in the 16S rRNA; (2) the reactivity patterns of the S15-16S rRNA and S15-fragment complexes are identical; (3) the protein protects defined RNA regions, located in the large interior loop and in the 3'-end strand of helix [655-672]-[734-751]; (4) the protein also causes enhanced chemical reactivity and enzyme accessibility interpreted as resulting from a local conformational rearrangement, induced by S15 binding. Images PMID:2453025

Vaccination with nontoxic mutant toxic shock syndrome toxin 1 protects against Staphylococcus aureus infection.

PubMed

Hu, Dong-Liang; Omoe, Katsuhiko; Sasaki, Sanae; Sashinami, Hiroshi; Sakuraba, Hirotake; Yokomizo, Yuichi; Shinagawa, Kunihiro; Nakane, Akio

2003-09-01

To investigate whether vaccination with nontoxic mutant toxic shock syndrome toxin 1 (mTSST-1) can protect against Staphylococcus aureus infection, mice were vaccinated with mTSST-1 and challenged with viable S. aureus. Survival in the mTSST-1-vaccinated group was higher, and bacterial counts in organs were significantly lower than those of control mice. Passive transfer of mTSST-1-specific antibodies also provided protection against S. aureus-induced septic death. Interferon (IFN)-gamma production in the serum samples and spleens from vaccinated mice was significantly decreased compared with that in controls, whereas interleukin-10 titers were significantly higher in vaccinated mice. IFN-gamma and tumor necrosis factor-alpha production in vitro were significantly inhibited by serum samples from mTSST-1-immunized mice but not from control mice. These results suggest that vaccination with mTSST-1 devoid of superantigenic properties provides protection against S. aureus infection and that the protection might be mediated by TSST-1-neutralizing antibodies as well as by the down-regulation of IFN-gamma production.

Dnmt1-dependent Chk1 pathway suppression is protective against neuron division.

PubMed

Oshikawa, Mio; Okada, Kei; Tabata, Hidenori; Nagata, Koh-Ichi; Ajioka, Itsuki

2017-09-15

Neuronal differentiation and cell-cycle exit are tightly coordinated, even in pathological situations. When pathological neurons re-enter the cell cycle and progress through the S phase, they undergo cell death instead of division. However, the mechanisms underlying mitotic resistance are mostly unknown. Here, we have found that acute inactivation of retinoblastoma (Rb) family proteins (Rb, p107 and p130) in mouse postmitotic neurons leads to cell death after S-phase progression. Checkpoint kinase 1 (Chk1) pathway activation during the S phase prevented the cell death, and allowed the division of cortical neurons that had undergone acute Rb family inactivation, oxygen-glucose deprivation (OGD) or in vivo hypoxia-ischemia. During neurogenesis, cortical neurons became protected from S-phase Chk1 pathway activation by the DNA methyltransferase Dnmt1, and underwent cell death after S-phase progression. Our results indicate that Chk1 pathway activation overrides mitotic safeguards and uncouples neuronal differentiation from mitotic resistance. © 2017. Published by The Company of Biologists Ltd.

[In vitro study of joint intervention of E-cad and Bmi-1 mediated by transcription activator-like effector nuclease in nasopharyngeal carcinoma].

PubMed

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 P<0.01).
 Conclusion: The joint intervention of E-cad 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.

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

PubMed Central

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

Zinc finger nuclease technology: advances and obstacles in modelling and treating genetic disorders.

PubMed

Jabalameli, Hamid Reza; Zahednasab, Hamid; Karimi-Moghaddam, Amin; Jabalameli, Mohammad Reza

2015-03-01

Zinc finger nucleases (ZFNs) are engineered restriction enzymes designed to target specific DNA sequences within the genome. Assembly of zinc finger DNA-binding domain to a DNA-cleavage domain enables the enzyme machinery to target unique locus in the genome and invoke endogenous DNA repair mechanisms. This machinery offers a versatile approach in allele editing and gene therapy. Here we discuss the architecture of ZFNs and strategies for generating targeted modifications within the genome. We review advances in gene therapy and modelling of the disease using these enzymes and finally, discuss the practical obstacles in using this technology. Copyright © 2014 Elsevier B.V. All rights reserved.

Proteases and nucleases involved in the biphasic digestion process of the brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae).

PubMed

Lomate, Purushottam R; Bonning, Bryony C

2018-07-01

Management of the brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), an invasive, agricultural pest in the United States, has presented significant challenges. This polyphagous insect uses both extra-oral and gut-based digestion thwarting protein- or nucleotide-based control strategies. The objective of this study was to biochemically characterize the digestive enzymes (proteases and nucleases) from the saliva, salivary gland and the gut of H. halys. Enzyme profiles for the two tissues and saliva radically differ: The pH optimum for proteases in the gut was six, with cysteine proteases predominant. In contrast, the alkaline pH optima for protease activity in the salivary gland (8-10) and saliva (7) reflected abundant serine protease and cathepsin activities. RNase enzymes were most abundant in saliva, while dsRNase and DNase activities were higher in the salivary gland and saliva compared to those in the gut. These very different enzyme profiles highlight the biphasic digestive system used by this invasive species for efficient processing of plant nutrients. Knowledge of H. halys digestive physiology will allow for counteractive measures targeting digestive enzymes or for appropriate protection of protein- or nucleotide-based management options targeting this pest. © 2018 Wiley Periodicals, Inc.

DNA targeting specificity of RNA-guided Cas9 nucleases.

PubMed

Hsu, Patrick D; Scott, David A; Weinstein, Joshua A; Ran, F Ann; Konermann, Silvana; Agarwala, Vineeta; Li, Yinqing; Fine, Eli J; Wu, Xuebing; Shalem, Ophir; Cradick, Thomas J; Marraffini, Luciano A; Bao, Gang; Zhang, Feng

2013-09-01

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding.

PubMed

Podevin, Nancy; Davies, Howard V; Hartung, Frank; Nogué, Fabien; Casacuberta, Josep M

2013-06-01

Conventional plant breeding exploits existing genetic variability and introduces new variability by mutagenesis. This has proven highly successful in securing food supplies for an ever-growing human population. The use of genetically modified plants is a complementary approach but all plant breeding techniques have limitations. Here, we discuss how the recent evolution of targeted mutagenesis and DNA insertion techniques based on tailor-made site-directed nucleases (SDNs) provides opportunities to overcome such limitations. Plant breeding companies are exploiting SDNs to develop a new generation of crops with new and improved traits. Nevertheless, some technical limitations as well as significant uncertainties on the regulatory status of SDNs may challenge their use for commercial plant breeding. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

PubMed Central

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

Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bach, Christian; Sherman, William; Pallis, Jani

Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations. This cytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with high affinity. In general, binding preferences of transcription factors are associated with significant degenerated diversity and complexity which convolutes the design and engineering of precise DNA binding domains. Evolutionary success of natural zinc finger proteins, however, evinces that nature created specific evolutionary traits and strategies, such as modularity and rank-specific recognition to cope with binding complexity that are critical for creating clinical viable toolsmore » to precisely modify the human genome. Our findings indicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-finger binding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger.« less

Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases

DOE PAGES

Bach, Christian; Sherman, William; Pallis, Jani; ...

2014-01-01

Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations. This cytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with high affinity. In general, binding preferences of transcription factors are associated with significant degenerated diversity and complexity which convolutes the design and engineering of precise DNA binding domains. Evolutionary success of natural zinc finger proteins, however, evinces that nature created specific evolutionary traits and strategies, such as modularity and rank-specific recognition to cope with binding complexity that are critical for creating clinical viable toolsmore » to precisely modify the human genome. Our findings indicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-finger binding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger.« less

26 CFR 1.7702B-1 - Consumer protection provisions.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 13 2011-04-01 2011-04-01 false Consumer protection provisions. 1.7702B-1 Section 1.7702B-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) General Actuarial Valuations § 1.7702B-1 Consumer protection...

Entirely S-protected chitosan: A promising mucoadhesive excipient for metronidazole vaginal tablets.

PubMed

Lupo, Noemi; Fodor, Benjamin; Muhammad, Ijaz; Yaqoob, Muhammad; Matuszczak, Barbara; Bernkop-Schnürch, Andreas

2017-12-01

Synthesis and evaluation of an entirely S-protected chitosan as mucoadhesive excipient for vaginal drug delivery. N-acetyl-cysteine was linked to 6-mercaptonicotinamide via disulphide exchange reaction. The obtained ligand, NAC-6-MNA, was subsequently attached to chitosan by carbodiimide mediated amide bond formation in two concentrations. The synthesized S-protected chitosan was chemically characterized and mucoadhesive properties and stability against oxidation were investigated. Moreover, metronidazole tablets comprising the S-protected chitosan were evaluated regarding water uptake capacity, disintegration behaviour, residence time on vaginal mucosa, release of the encapsulated drug and antimicrobial activity. S-protected chitosan displayed 160±19 (CS-MNA-160) and 320±38 (CS-MNA-320)µmol of ligand per gram of polymer. At pH 4.2, CS-MNA-160 and CS-MNA-320 showed 5.2-fold and 6.2-fold increase in mucus viscosity in comparison to unmodified chitosan (One-way ANOVA, p<.001), whereas, 9.9-fold (CS-MNA-160) and 15.6-fold (CS-MNA-320) (One-way ANOVA, p<.001) increase in viscosity was measured at pH 6. The S-protected chitosan remained stable against oxidation in presence of 0.5%v/v hydrogen peroxide. Metronidazole tablets consisting in S-protected chitosan showed prolonged residence time on vaginal mucosa and improved water uptake capacity and disintegration time in comparison to tablets consisting of unmodified chitosan. Moreover, CS-MNA-320 metronidazole tablets displayed prolonged drug release and antimicrobial activity. On the basis of the achieved results, entirely S-protected chitosan represents a promising excipient for the development of metronidazole vaginal tablets. S-protected thiomers are polymers modified with thiol groups protected by aromatic ligands and characterized by strong mucoadhesive properties and high stability against oxidation. Up to date, the entirely S-protection of thiol groups was achieved via the synthesis of the ligand 2-((2-amino-2

G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding

PubMed Central

Ray, Sujay; Bandaria, Jigar N.; Qureshi, Mohammad H.; Yildiz, Ahmet; Balci, Hamza

2014-01-01

Human telomeres terminate with a single-stranded 3′ G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). The protection of telomeres (POT1)/POT1-interacting protein 1 (TPP1) heterodimer binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K+, POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA’s access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na+, in which ssTEL forms only a less-stable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals. PMID:24516170

S2 expressed from recombinant virus confers broad protection against infectious bronchitis virus

USDA-ARS?s Scientific Manuscript database

We previously demonstrated that overexposing the IBV (infectious bronchitis virus) S2 to the chicken immune system by means of a vectored vaccine, followed by boost with whole virus, protects chickens against IBV showing dissimilar S1. We developed recombinant Newcastle disease virus (NDV) LaSota (...

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

PubMed

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.

22 CFR 123.6 - Foreign trade zones and U.S. Customs and Border Protection bonded warehouses.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 22 Foreign Relations 1 2013-04-01 2013-04-01 false Foreign trade zones and U.S. Customs and Border Protection bonded warehouses. 123.6 Section 123.6 Foreign Relations DEPARTMENT OF STATE INTERNATIONAL TRAFFIC IN ARMS REGULATIONS LICENSES FOR THE EXPORT OF DEFENSE ARTICLES § 123.6 Foreign trade zones and U.S. Customs and Border Protection bonded...

Degradation of recombinant proteins by CHO host cell proteases is prevented by Matriptase-1 knock-out.

PubMed

Laux, Holger; Romand, Sandrine; Nuciforo, Sandro; Farady, Christopher J; Tapparel, Joel; Buechmann-Moeller, Stine; Sommer, Benjamin; Oakeley, Edward J; Bodendorf, Ursula

2018-05-19

An increasing number of non-antibody format proteins are entering the clinical development. However, one of the major hurdles for the production of non-antibody glycoproteins is host cell-related proteolytic degradation, which can drastically impact developability and timelines of pipeline projects. Chinese hamster ovary (CHO) cells are the preferred production host for recombinant therapeutic proteins. Using protease inhibitors, transcriptomics and genetic knockdowns we have identified, out of the more than 700 known proteases in rodents, Matriptase-1 as the major protease involved in degradation of recombinant proteins expressed in CHO-K1 cells. Subsequently Matriptase-1 was deleted in CHO-K1 cells using "Transcription Activator-Like Effector Nucleases" (TALENs) as well as zinc-finger nucleases (ZFNs). This resulted in a superior CHO-K1 matriptase knockout (KO) cell line with strongly reduced or no proteolytic degradation activity towards a panel of recombinantly-expressed proteins. The matriptase KO cell line was evaluated in spike-in experiments, and showed little or no degradation of proteins incubated in culture supernatant derived from the KO cells. This effect was confirmed when the same proteins were recombinantly expressed in the KO cell line. In summary, the combination of novel cell line engineering tools, next generation sequencing screening methods and the recently published Chinese hamster genome has enabled the development of this novel matriptase KO CHO cell line capable of improving expression yields of intact therapeutic proteins. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Innate Immune Dysfunctions in Aged Mice Facilitate the Systemic Dissemination of Methicillin-Resistant S. aureus

PubMed Central

Tseng, Ching Wen; Kyme, Pierre A.; Arruda, Andrea; Ramanujan, V. Krishnan; Tawackoli, Wafa; Liu, George Y.

2012-01-01

Elderly humans show increased susceptibility to invasive staphylococcal disease after skin and soft tissue infection. However, it is not understood how host immunity changes with aging, and how that predisposes to invasive disease. In a model of severe skin infection, we showed that aged mice (16- to 20-month-old) exhibit dramatic bacterial dissemination compared with young adult mice (2-month-old). Bacterial dissemination was associated with significant reductions of CXCL1 (KC), polymorphonuclear cells (PMNs), and extracellular DNA traps (NETs) at the infection site. PMNs and primary skin fibroblasts isolated from aged mice showed decreased secretion of CXCL2 (MIP-2) and KC in response to MRSA, and in vitro analyses of mitochondrial functions revealed that the mitochondrial electron transport chain complex I plays a significant role in induction of chemokines in the cells isolated from young but not old mice. Additionally, PMNs isolated from aged mice have reduced ability to form NETs and to kill MRSA. Expression of nuclease by S. aureus led to increased bacterial systemic dissemination in young but not old mice, suggesting that defective NETs formation in elderly mice permitted nuclease and non-nuclease expressing S. aureus to disseminate equally well. Overall, these findings suggest that gross impairment of both skin barrier function and innate immunity contributes to the propensity for MRSA to disseminate in aged mice. Furthermore, the study indicates that contribution of bacterial factors to pathogenicity may vary with host age. PMID:22844481

Inhibition of aac(6′)-Ib-mediated amikacin resistance by nuclease-resistant external guide sequences in bacteria

PubMed Central

Soler Bistué, Alfonso J. C.; Martín, Fernando A.; Vozza, Nicolás; Ha, Hongphuc; Joaquín, Jonathan C.; Zorreguieta, Angeles; Tolmasky, Marcelo E.

2009-01-01

Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6′)-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6′)-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nuclease-resistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6′)-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect. PMID:19666539

A recombinant fowl adenovirus expressing the S1 gene of infectious bronchitis virus protects against challenge with infectious bronchitis virus.

PubMed

Johnson, Michael A; Pooley, Catherine; Ignjatovic, Jagoda; Tyack, Scott G

2003-06-20

The spike peplomer S1 subunit sequence from avian infectious bronchitis virus (IBV) Vic S strain was expressed in a plasmid under the control of the fowl adenovirus (FAV) major late promoter (MLP). Two recombinants were constructed in FAV serotype 8 (FAV 8) by inserting the expression cassette between the SnaBI and XbaI restriction enzyme sites (clone DA3) or between the SpeI sites (clone CA6-20). Expression of the S1 gene in the recombinants was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) by 20h post-infection. Commercial broiler chickens were orally vaccinated at day 0 or day 6 post-hatch and challenged at day 35 post-hatch. FAV antibody ELISA confirmed that maternal antibody directed against inclusion body hepatitis (serotype 8) had decayed in control birds and that FAV specific serum IgG responses were produced in vaccinated birds at the time of challenge. Further, an S1 specific antibody response was detected prior to challenge. Birds were challenged with either Vic S (serotype B) or N1/62 (serotype C) strains of IBV. The tracheas of challenged birds were analyzed by RT-PCR and re-isolation of virus. In birds vaccinated at day 6, 90-100% protection at the trachea was induced against either homologous or heterologous challenge. The construction of a recombinant FAV expressing S1 of IBV demonstrates the potential of an alternative vaccination strategy against IBV.

Ribosomal protein S6 kinase 1 signaling regulates mammalian lifespan

PubMed Central

Selman, Colin; Tullet, Jennifer M.A.; Wieser, Daniela; Irvine, Elaine; Lingard, Steven J.; Choudhury, Agharul I.; Claret, Marc; Al-Qassab, Hind; Carmignac, Danielle; Ramadani, Faruk; Woods, Angela; Robinson, Iain C.A.; Schuster, Eugene; Batterham, Rachel L.; Kozma, Sara C.; Thomas, George; Carling, David; Okkenhaug, Klaus; Thornton, Janet M.; Partridge, Linda; Gems, David; Withers, Dominic J.

2016-01-01

Caloric restriction (CR) protects against aging and disease but the mechanisms by which this affects mammalian lifespan are unclear. We show in mice that deletion of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway component ribosomal S6 protein kinase 1 (S6K1) led to increased lifespan and resistance to age-related pathologies such as bone, immune and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian lifespan, and suggest therapeutic manipulation of S6K1 and AMPK might mimic CR and provide broad protection against diseases of aging. PMID:19797661

Stress-induced NQO1 controls stability of C/EBPα against 20S proteasomal degradation to regulate p63 expression with implications in protection against chemical-induced skin cancer.

PubMed

Patrick, B A; Jaiswal, A K

2012-10-04

Previously, we have shown a role of cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) in the stabilization of p63 against 20S proteasomal degradation resulting in thinning of the epithelium and chemical-induced skin cancer (Oncogene (2011) 30, 1098-1107). Current studies have demonstrated that NQO1 control of CCAAT-enhancer binding protein (C/EBPα) against 20S proteasomal degradation also contributes to the upregulation of p63 expression and protection. Western and immunohistochemistry analysis revealed that disruption of the NQO1 gene in mice and mouse keratinocytes led to degradation of C/EBPα and loss of p63 gene expression. p63 promoter mutagenesis, transfection and chromatin immunoprecipitation assays identified a C/EBPα-binding site between nucleotide position -185 and -174 that bound to C/EBPα and upregulated p63 gene expression. Co-immunoprecipitation and immunoblot analysis demonstrated that 20S proteasomes directly interacted and degraded C/EBPα. NQO1 direct interaction with C/EBPα led to stabilization of C/EBPα against 20S proteasomal degradation. NQO1 protection of C/EBPα required binding of NADH with NQO1. Exposure of skin and keratinocytes to the chemical stress agent benzo(a)pyrene led to induction of NQO1 and stabilization of C/EBPα protein, resulting in an increase in p63 RNA and protein in wild-type but not in NQO1-/- mice. Collectively, the current data combined with previous data suggest that stress induction of NQO1 through both stabilization of C/EBPα and increase in p63 and direct stabilization of p63 controls keratinocyte differentiation, leading to protection against chemical-induced skin carcinogenesis. The studies are significant as 2-4% human individuals are homozygous and 23% are heterozygous for the NQO1P187S mutation and might be susceptible to stress-induced skin diseases.

MCCE analysis of the pKas of introduced buried acids and bases in staphylococcal nuclease.

PubMed

Gunner, M R; Zhu, Xuyu; Klein, Max C

2011-12-01

The pK(a)s of 96 acids and bases introduced into buried sites in the staphylococcal nuclease protein (SNase) were calculated using the multiconformation continuum electrostatics (MCCE) program and the results compared with experimental values. The pK(a)s are obtained by Monte Carlo sampling of coupled side chain protonation and position as a function of pH. The dependence of the results on the protein dielectric constant (ε(prot)) in the continuum electrostatics analysis and on the Lennard-Jones non-electrostatics parameters was evaluated. The pK(a)s of the introduced residues have a clear dependence on ε(prot,) whereas native ionizable residues do not. The native residues have electrostatic interactions with other residues in the protein favoring ionization, which are larger than the desolvation penalty favoring the neutral state. Increasing ε(prot) scales both terms, which for these residues leads to small changes in pK(a). The introduced residues have a larger desolvation penalty and negligible interactions with residues in the protein. For these residues, changing ε(prot) has a large influence on the calculated pK(a). An ε(prot) of 8-10 and a Lennard-Jones scaling of 0.25 is best here. The X-ray crystal structures of the mutated proteins are found to provide somewhat better results than calculations carried out on mutations made in silico. Initial relaxation of the in silico mutations by Gromacs and extensive side chain rotamer sampling within MCCE can significantly improve the match with experiment. Copyright © 2011 Wiley-Liss, Inc.

Ten Years of Addressing Children’s Health through Regulatory Policy at the U.S. Environmental Protection Agency

PubMed Central

Payne-Sturges, Devon; Kemp, Debra

2008-01-01

Background Executive Order (EO) 13045, Protection of Children From Environmental Health Risks and Safety Risks, directs each federal agency to ensure that its policies, programs, activities, and standards address disproportionate environmental health and safety risks to children. Objectives We reviewed regulatory actions published by U.S. Environmental Protection Agency (EPA) in the Federal Register from April 1998 through December 2006 to evaluate applicability of EO 13045 to U.S. EPA actions and consideration of children’s health issues in U.S. EPA rulemakings. Discussion Although virtually all actions discussed EO 13045, fewer than two regulations per year, on average, were subject to the EO requirement to evaluate children’s environmental health risks. Nonetheless, U.S. EPA considered children’s environmental health in all actions addressing health or safety risks that may disproportionately affect children. Conclusion The EO does not apply to a broad enough set of regulatory actions to ensure protection of children’s health and safety risks, largely because of the small number of rules that are economically significant. However, given the large number of regulations that consider children’s health issues despite not being subject to the EO, other statutory requirements and agency policies reach a larger set of regulations to ensure protection of children’s environmental health. PMID:19079726

S-Enantiomer of 19-Hydroxyeicosatetraenoic Acid Preferentially Protects Against Angiotensin II-Induced Cardiac Hypertrophy.

PubMed

Shoieb, Sherif M; El-Kadi, Ayman O S

2018-06-07

We have recently demonstrated that the racemic mixture of 19-hydroxyeicosatetraenoic acid (19-HETE) protects against angiotensin II (Ang II) induced cardiac hypertrophy. Therefore, the purpose of this study was to investigate whether R- or S-enantiomer of 19-HETE confers cardioprotection against Ang II-induced cellular hypertrophy in RL-14 and H9c2 cells. Both cell lines were treated with vehicle or 10 μM Ang II in the absence and presence of 20 &#[mu]M 19(R)-HETE or 19(S)-HETE for 24 h. Thereafter, the level of mid-chain HETEs was determined using liquid chromatography - mass spectrometry (LC/MS). Gene and protein expression levels were measured using real-time PCR and Western blot analysis, respectively. The results showed that both 19(R)-HETE and 19(S)-HETE significantly decreased the metabolite formation rate of midchain HETEs namely 8-, 9-, 12- and 15-HETE compared to control group while the level of 5-HETE was selectively decreased by S-enantiomer. Moreover, both 19(R)-HETE and 19(S)-HETE significantly inhibited the catalytic activity of CYP1B1 and decreased the protein expression of 5- and 12-lipoxxygenase (LOX) as well as cyclooxygenase-2 (COX-2). Notably, the decrease in 15-LOX protein expression was only mediated by 19(S)-HETE. Moreover, both enantiomers protected against Ang II-induced cellular hypertrophy as evidenced by a significant decrease in mRNA expression of β/α-myosin heavy chain ratio, ANP, IL-6 and IL-8. Our data demonstrated that S-enantiomer of 19-HETE preferentially protected against Ang II-induced cellular hypertrophy via decreasing the level of mid-chain HETEs, inhibiting catalytic activity of CYP1B1, decreasing protein expression of LOX and COX-2 enzymes and decreasing mRNA expression of IL-6 and IL-8. The American Society for Pharmacology and Experimental Therapeutics.

Sensitive detection of microRNAs based on the conversion of colorimetric assay into electrochemical analysis with duplex-specific nuclease-assisted signal amplification

PubMed Central

Xia, Ning; Liu, Ke; Zhou, Yingying; Li, Yuanyuan; Yi, Xinyao

2017-01-01

miRNAs have emerged as new biomarkers for the detection of a wide variety of cancers. By employing duplex-specific nuclease for signal amplification and gold nanoparticles (AuNPs) as the carriers of detection probes, a novel electrochemical assay of miRNAs was performed. The method is based on conversion of the well-known colorimetric assay into electrochemical analysis with enhanced sensitivity. DNA capture probes immobilized on the electrode surface and ferrocene (Fc)-labeled DNA detection probes (denoted “Fc-DNA-Fc”) presented in the solution induced the assembly of positively charged AuNPs on the electrode surface through the electrostatic interaction. As a result, a large number of Fc-DNA-Fc molecules were attached on the electrode surface, thus amplifying the electrochemical signal. When duplex-specific nuclease was added to recycle the process of miRNA-initiated digestion of the immobilized DNA probes, Fc-DNA-Fc-induced assembly of AuNPs on the electrode surface could not occur. This resulted in a significant fall in the oxidation current of Fc. The current was found to be inversely proportional to the concentration of miRNAs in the range of 0–25 fM, and a detection limit of 0.1 fM was achieved. Moreover, this work presents a new method for converting colorimetric assays into sensitive electrochemical analyses, and thus would be valuable for design of novel chemical/biosensors. PMID:28761341

Zinc finger nuclease-mediated precision genome editing of an endogenous gene in hexaploid bread wheat (Triticum aestivum) using a DNA repair template.

PubMed

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.

Syntheses of prodrug-type phosphotriester oligonucleotides responsive to intracellular reducing environment for improvement of cell membrane permeability and nuclease resistance.

PubMed

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.

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

PubMed

Glazkova, D V; Shipulin, G A

2014-01-01

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

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

PubMed Central

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

DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1

EPA Science Inventory

DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1. R A Pegram1 and M K Ross2. 2Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC; 1Pharmacokinetics Branch, NHEERL, ORD, United States Environmental Protection Ag...

Wld S protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice.

PubMed

Conforti, Laura; Wilbrey, Anna; Morreale, Giacomo; Janeckova, Lucie; Beirowski, Bogdan; Adalbert, Robert; Mazzola, Francesca; Di Stefano, Michele; Hartley, Robert; Babetto, Elisabetta; Smith, Trevor; Gilley, Jonathan; Billington, Richard A; Genazzani, Armando A; Ribchester, Richard R; Magni, Giulio; Coleman, Michael

2009-02-23

The slow Wallerian degeneration (Wld(S)) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70-amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide-synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of Wld(S)-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the Wld(S) VCP-binding domain with an alternative ataxin-3-derived VCP-binding sequence restores its protective function. Enzyme-dead Wld(S) is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. Wld(S) requires both of its components to protect axons from degeneration.

Asymmetric binding of histone H1 stabilizes MMTV nucleosomes and the interaction of progesterone receptor with the exposed HRE.

PubMed

Vicent, Guillermo P; Meliá, María J; Beato, Miguel

2002-11-29

Packaging of mouse mammary tumor virus (MMTV) promoter sequences in nucleosomes modulates access of DNA binding proteins and influences the interaction among DNA bound transcription factors. Here we analyze the binding of histone H1 to MMTV mononucleosomes assembled with recombinant histones and study its influence on nucleosome structure and stability as well as on progesterone receptor (PR) binding to the hormone responsive elements (HREs). The MMTV nucleosomes can be separated into three main populations, two of which exhibited precise translational positioning. Histone H1 bound preferentially to the 5' distal nucleosomal DNA protecting additional 27-28 nt from digestion by micrococcal nuclease. Binding of histone H1 was unaffected by prior crosslinking of protein and DNA in nucleosomes with formaldehyde. Neither the translational nor the rotational nucleosome positioning was altered by histone H1 binding, but the nucleosomes were stabilized as judged by the kinetics of nuclease cleavage. Unexpectedly, binding of recombinant PR to the exposed distal HRE-I in nucleosomes was enhanced in the presence of histone H1, as demonstrated by band shift and footprinting experiments. This enhanced PR affinity may contribute to the reported positive effect of histone H1 on the hormonal activation of MMTV reporter genes.

Research at the U.S. EPA’s Ground Water and Ecosystems Protection Division

EPA Science Inventory

The U.S. EPA’s Office of Research and Development (ORD) conducts leading-edge research and fosters the sound use of science and technology to fulfill the Agency's mission to protect human health and safeguard the natural environment. The mission of the Ground Water and Ecosystem...

S1PR3 is essential for phosphorylated fingolimod to protect astrocytes against oxygen-glucose deprivation-induced neuroinflammation via inhibiting TLR2/4-NFκB signalling.

PubMed

Dong, Yin-Feng; Guo, Ruo-Bing; Ji, Juan; Cao, Lu-Lu; Zhang, Ling; Chen, Zheng-Zhen; Huang, Ji-Ye; Wu, Jin; Lu, Jun; Sun, Xiu-Lan

2018-03-13

Fingolimod (FTY720) is used as an immunosuppressant for multiple sclerosis. Numerous studies indicated its neuroprotective effects in stroke. However, the mechanism remains to be elucidated. This study was intended to investigate the mechanisms of phosphorylated FTY720 (pFTY720), which was the principle active molecule in regulating astrocyte-mediated inflammatory responses induced by oxygen-glucose deprivation (OGD). Results demonstrated that pFTY720 could protect astrocytes against OGD-induced injury and inflammatory responses. It significantly decreased pro-inflammatory cytokines, including high mobility group box 1 (HMGB1) and tumour necrosis factor-α (TNF-α). Further, studies displayed that pFTY720 could prevent up-regulation of Toll-like receptor 2 (TLR2), phosphorylation of phosphoinositide 3-kinase (PI3K) and nuclear translocation of nuclear factor kappa B (NFκB) p65 subunit caused by OGD. Sphingosine-1-phosphate receptor 3 (S1PR3) knockdown could reverse the above change. Moreover, administration of TLR2/4 blocker abolished the protective effects of pFTY720. Taken together, this study reveals that pFTY720 depends on S1PR3 to protect astrocytes against OGD-induced neuroinflammation, due to inhibiting TLR2/4-PI3K-NFκB signalling pathway. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Protein S is protective in pulmonary fibrosis.

PubMed

Urawa, M; Kobayashi, T; D'Alessandro-Gabazza, C N; Fujimoto, H; Toda, M; Roeen, Z; Hinneh, J A; Yasuma, T; Takei, Y; Taguchi, O; Gabazza, E C

2016-08-01

Essentials Epithelial cell apoptosis is critical in the pathogenesis of idiopathic pulmonary fibrosis. Protein S, a circulating anticoagulant, inhibited apoptosis of lung epithelial cells. Overexpression of protein S in lung cells reduced bleomycin-induced pulmonary fibrosis. Intranasal therapy with exogenous protein S ameliorated bleomycin-induced pulmonary fibrosis. Background Pulmonary fibrosis is the terminal stage of interstitial lung diseases, some of them being incurable and of unknown etiology. Apoptosis plays a critical role in lung fibrogenesis. Protein S is a plasma anticoagulant with potent antiapoptotic activity. The role of protein S in pulmonary fibrosis is unknown. Objectives To evaluate the clinical relevance of protein S and its protective role in pulmonary fibrosis. Methods and Results The circulating level of protein S was measured in patients with pulmonary fibrosis and controls by the use of enzyme immunoassays. Pulmonary fibrosis was induced with bleomycin in transgenic mice overexpressing human protein S and wild-type mice, and exogenous protein S or vehicle was administered to wild-type mice; fibrosis was then compared in both models. Patients with pulmonary fibrosis had reduced circulating levels of protein S as compared with controls. Inflammatory changes, the levels of profibrotic cytokines, fibrosis score, hydroxyproline content in the lungs and oxygen desaturation were significantly reduced in protein S-transgenic mice as compared with wild-type mice. Wild-type mice treated with exogenous protein S showed significant decreases in the levels of inflammatory and profibrotic markers and fibrosis in the lungs as compared with untreated control mice. After bleomycin infusion, mice overexpressing human protein S showed significantly low caspase-3 activity, enhanced expression of antiapoptotic molecules and enhanced Akt and Axl kinase phosphorylation as compared with wild-type counterparts. Protein S also inhibited apoptosis of alveolar

Force Protection Leader’s Guide

DTIC Science & Technology

1993-01-01

and TC1-13, Hot Weather Flying Sense. • As a minimum: □ Taxiing. Get helicopter airborne and through ETL as quickly as possible to rninimize sand...protective covers between flights to protect aircraft from excess heat and to stop sand and dirt from getting into moving parts. • Wipe oil and grease...nonabrasive material—then attach cover. • Add oil and hydraulic fluid directly from original unopened containers to help stop sand and dirt from getting

Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor

PubMed Central

2010-01-01

Background Staphylococcal (or micrococcal) nuclease or thermonuclease (SNase or Nuc) is a naturally-secreted nucleic acid degrading enzyme that participates in Staphylococcus aureus spread in the infected host. Purified Nuc protein can be used as an exogenous reagent to clear cellular extracts and improve protein purification. Here, a recombinant form of Nuc was produced and secreted in a Gram-positive host, Lactococcus lactis, and purified from the culture medium. Results The gene segment corresponding to the S. aureus nuclease without its signal peptide was cloned in an expression-secretion vector. It was then fused to a lactococcal sequence encoding a signal peptide, and expressed under the control of a lactococcal promoter that is inducible by zinc starvation. An L. lactis subsp cremoris model strain (MG1363) transformed with the resulting plasmid was grown in either of two media (GM17v and CDM) that are free of animal compounds, allowing GMP (Good Manufacturing Practice) production. Induction conditions (concentration of the metal chelator EDTA and timing of addition) in small-scale pH-regulated fermentors were optimized using LacMF (Lactis Multi-Fermentor), a home-made parallel fermentation control system able to monitor 12 reactors simultaneously. Large amounts of recombinant Nuc (rNuc) were produced and secreted in both media, and rNuc was purified from GM17v medium in a single-step procedure. Conclusions In L. lactis, rNuc production and secretion were optimal after induction by 0.5 mM EDTA in small scale (200 mL) GM17v exponential phase cultures (at an OD600 of 2), leading to a maximal protein yield of 210 mg per L of culture medium. Purified rNuc was highly active, displaying a specific activity of 2000 U/mg. PMID:20492646

Synthesis and in vitro characterization of entirely S-protected thiolated pectin for drug delivery.

PubMed

Hintzen, Fabian; Hauptstein, Sabine; Perera, Glen; Bernkop-Schnürch, Andreas

2013-11-01

The study was aimed to synthesize a thiolated polymer (thiomer) that is resistant to oxidation in solutions above pH 5. In order to protect a pectin-cysteine conjugate against premature oxidation, the thiomer was S-protected by a disulfide connected leaving group. Therefore, 2-mercaptonicotinic acid was first coupled to L-cysteine by a disulfide exchange reaction and the purified product was subsequently attached to pectin by a carbodiimide mediated amid bond formation. The obtained fully S-protected thiolated pectin was in vitro characterized with respect to co- and mucoadhesive properties and stability toward oxidation. The results indicated a 1.8-fold and 2.3-fold enhanced disintegration time at pH 6.8 of the S-protected thiolated pectin (Pec-Cys-MNA) compared to thiolated pectin (Pec-Cys) and unmodified pectin (Pec). Moreover, rheological measurements of polymer/mucus mixtures showed a 1.6-fold (compared to Pec-Cys) and 6.7-fold (compared to Pec) increased dynamic viscosity of Pec-Cys-MNA. On the other hand, in the presence of a strong oxidizing agent such as H2O2 (0.3% v/v), no increase in viscosity of Pec-Cys-MNA could be observed. A 6-month experiment also demonstrated the long-term stability of a liquid formulation based on Pec-Cys-MNA. Further investigations proved that the first time all thiol groups on a thiolated polymer could be protected owing to the novel synthesis. Accordingly, these features may help to develop thiomer based liquid or gel formulations targeting mucosal surfaces such as nasal, ocular or vaginal drug delivery systems. Copyright © 2013 Elsevier B.V. All rights reserved.

Critical Foundations: Protecting America’s Infrastructures. The Report of the President’s Commission on Critical Infrastructure Protection.

DTIC Science & Technology

1997-10-13

in Tokyo and bombings of the subway system in Paris, mass transit remains open and vulnerable to terrorist acts. Millions of miles of pipelines carry...Outside of "" C Risk Management Plan . Creates a Competitive Advantage , Not Considered Industry’s "• Protects Brand/ Franchise Responsibility- I ssue of...the out- set, the Commission considered expanding the scope of this sector to include food , health care and the nation’s work force as additional

Targeting Sirna Missiles to Her2+ Breast Cancer

DTIC Science & Technology

2008-06-01

intact and appears to be protected from serum nucleases (Fig. 1) . T7 -transcribed siRNA induces higher breast cancer cell cytotoxicity than synthetic...cytotoxicity of T7 transcribed vs s y n t h e t i c anti-HER2 siRNA on HER2+ cells. We acquired a 21 nucleotide (nt) s y n t h e t i c anti-HER2...ErbB2) siRNA and also produced a T7 -transcribed molecule (Silencer Principal Investigator: Medina-Kauwe, Lali K. 2 siRNA construction kit; Ambion) using

H2S protects against methionine-induced oxidative stress in brain endothelial cells.

PubMed

Tyagi, Neetu; Moshal, Karni S; Sen, Utpal; Vacek, Thomas P; Kumar, Munish; Hughes, William M; Kundu, Soumi; Tyagi, Suresh C

2009-01-01

Homocysteine (Hcy) causes cerebrovascular dysfunction by inducing oxidative stress. However, to date, there are no strategies to prevent Hcy-induced oxidative damage. Hcy is an H2S precursor formed from methionine (Met) metabolism. We aimed to investigate whether H2S ameliorated Met-induced oxidative stress in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to Met treatment in the presence or absence of NaHS (donor of H2S). Met-induced cell toxicity increased the levels of free radicals in a concentration-dependent manner. Met increased NADPH-oxidase-4 (NOX-4) expression and mitigated thioredxion-1(Trx-1) expression. Pretreatment of bEnd3 with NaHS (0.05 mM) attenuated the production of free radicals in the presence of Met and protected the cells from oxidative damage. Furthermore, NaHS enhanced inhibitory effects of apocynin, N-acetyl-l-cysteine (NAC), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), Nomega-nitro-l-arginine methyl ester (L-NAME) on ROS production and redox enzymes levels induced by Met. In conclusion, the administration of H2S protected the cells from oxidative stress induced by hyperhomocysteinemia (HHcy), which suggested that NaHS/H2S may have therapeutic potential against Met-induced oxidative stress.

From classical mutagenesis to nuclease-based breeding - directing natural DNA repair for a natural end-product.

PubMed

Pacher, Michael; Puchta, Holger

2017-05-01

Production of mutants of crop plants by the use of chemical or physical genotoxins has a long tradition. These factors induce the natural DNA repair machinery to repair damage in an error-prone way. In the case of radiation, multiple double-strand breaks (DSBs) are induced randomly in the genome, leading in very rare cases to a desirable phenotype. In recent years the use of synthetic, site-directed nucleases (SDNs) - also referred to as sequence-specific nucleases - like the CRISPR/Cas system has enabled scientists to use exactly the same naturally occurring DNA repair mechanisms for the controlled induction of genomic changes at pre-defined sites in plant genomes. As these changes are not necessarily associated with the permanent integration of foreign DNA, the obtained organisms per se cannot be regarded as genetically modified as there is no way to distinguish them from natural variants. This applies to changes induced by DSBs as well as single-strand breaks, and involves repair by non-homologous end-joining and homologous recombination. The recent development of SDN-based 'DNA-free' approaches makes mutagenesis strategies in classical breeding indistinguishable from SDN-derived targeted genome modifications, even in regard to current regulatory rules. With the advent of new SDN technologies, much faster and more precise genome editing becomes available at reasonable cost, and potentially without requiring time-consuming deregulation of newly created phenotypes. This review will focus on classical mutagenesis breeding and the application of newly developed SDNs in order to emphasize similarities in the context of the regulatory situation for genetically modified crop plants. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Identifying and structuring objectives for a coral reef protection plan at the U.S. Environmental Protection Agency

EPA Science Inventory

Region 2 of the U.S. Environmental Protection Agency initiated a Coral Reef Protection Plan (CRPP) in 2014 to reduce anthropogenic stress on Caribbean coral reefs. The CRPP is intended to foster institutional practices that improve reef condition and focus regulatory and non-regu...

A tailored biocatalyst achieved by the rational anchoring of imidazole groups on a natural polymer: furnishing a potential artificial nuclease by sustainable materials engineering.

PubMed

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.

Human ribosomal RNA gene: nucleotide sequence of the transcription initiation region and comparison of three mammalian genes.

PubMed Central

Financsek, I; Mizumoto, K; Mishima, Y; Muramatsu, M

1982-01-01

The transcription initiation site of the human ribosomal RNA gene (rDNA) was located by using the single-strand specific nuclease protection method and by determining the first nucleotide of the in vitro capped 45S preribosomal RNA. The sequence of 1,211 nucleotides surrounding the initiation site was determined. The sequenced region was found to consist of 75% G and C and to contain a number of short direct and inverted repeats and palindromes. By comparison of the corresponding initiation regions of three mammalian species, several conserved sequences were found upstream and downstream from the transcription starting point. Two short A + T-rich sequences are present on human, mouse, and rat ribosomal RNA genes between the initiation site and 40 nucleotides upstream, and a C + T cluster is located at a position around -60. At and downstream from the initiation site, a common sequence, T-AG-C-T-G-A-C-A-C-G-C-T-G-T-C-C-T-CT-T, was found in the three genes from position -1 through +18. The strong conservation of these sequences suggests their functional significance in rDNA. The S1 nuclease protection experiments with cloned rDNA fragments indicated the presence in human 45S RNA of molecules several hundred nucleotides shorter than the supposed primary transcript. The first 19 nucleotides of these molecules appear identical--except for one mismatch--to the nucleotide sequence of the 5' end of a supposed early processing product of the mouse 45S RNA. Images PMID:6954460

Myostatin knockout using zinc-finger nucleases promotes proliferation of ovine primary satellite cells in vitro.

PubMed

Salabi, Fatemeh; Nazari, Mahmood; Chen, Qing; Nimal, Jonathan; Tong, Jianming; Cao, Wen G

2014-12-20

Myostatin (MSTN) has previously been shown to negatively regulate the proliferation and differentiation of skeletal muscle cells. Satellite cells are quiescent muscle stem cells that promote muscle growth and repair. Because the mechanism of MSTN in the biology of satellite cells is not well understood, this study was conducted to generate MSTN mono-allelic knockout satellite cells using the zinc-finger nuclease mRNA (MSTN-KO ZFN mRNA) and also to investigate the effect of this disruption on the proliferation and differentiation of sheep primary satellite cells (PSCs). Nineteen biallelic and four mono-allelic knockout cell clones were obtained after sequence analysis. The homologous mono-allelic knockout cells with 5-bp deletion were used to further evaluations. The results demonstrated that mono-allelic knockout of MSTN gene leads to translation inhibition. Real-time quantitative PCR results indicated that knockout of MSTN contributed to an increase in CDK2 and follistatin and a decrease in p21 at the transcript level in proliferation conditions. Moreover, MSTN knockout significantly increased the proliferation of mutant clones (P < 0.01). Consistent with the observed increase in CDK2 and decrease in p21 in cells lacking MSTN, cell cycle analysis showed that MSTN negatively regulated the G1 to S progression. In addition, knockout of myostatin resulted in a remarkable increase in MyoD and MyoG expression under differentiating conditions but had no effect on Myf5 expression. These results expanded our understanding of the regulation mechanism of MSTN. Furthermore, the MSTN-KO ZFN mRNA system in PSCs could be used to generate transgenic sheep in the future.

Orion’s protective backshell installed on This Week @NASA - August 25, 2014

NASA Image and Video Library

2014-08-25

Engineers at Kennedy Space Center have finished installing the Orion spacecraft’s backshell – the black protective tiles on the cone-shaped sides of NASA’s new deep space capsule. The backshell tiles are the same type that protected the underside of space shuttles -- and will not only provide protection from debris while in space but from extreme temperatures in that area of the spacecraft as it returns from space – which could exceed 31-hundred degrees Fahrenheit. Also, SLS anti-geyser testing, Webb’s replica backplane, Arctic Sea ice loss, Ancient Earth, Alien Earths and more!

LAST, a c-Myc-inducible long noncoding RNA, cooperates with CNBP to promote CCND1 mRNA stability in human cells

PubMed Central

Li, Jinming

2017-01-01

Cyclin D1 is a critical regulator of cell cycle progression and works at the G1 to S-phase transition. Here, we report the isolation and characterization of the novel c-Myc-regulated lncRNA LAST (LncRNA-Assisted Stabilization of Transcripts), which acts as a CCND1 mRNA stabilizer. Mechanistically, LAST was shown to cooperate with CNBP to bind to the 5′UTR of CCND1 mRNA to protect against possible nuclease targeting. In addition, data from CNBP RIP-seq and LAST RNA-seq showed that CCND1 mRNA might not be the only target of LAST and CNBP; three additional mRNAs were shown to be post-transcriptional targets of LAST and CNBP. In a xenograft model, depletion of LAST diminished and ectopic expression of LAST induced tumor formation, which are suggestive of its oncogenic function. We thus report a previously unknown lncRNA involved in the fine-tuned regulation of CCND1 mRNA stability, without which CCND1 exhibits, at most, partial expression. PMID:29199958

40 CFR 180.1281 - S-Abscisic Acid, (S)-5-(1-hydroxy-2,6,6-trimethyl-4-oxo-1-cyclohex-2-enyl)-3-methyl-penta-(2Z,4E...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false S-Abscisic Acid, (S)-5-(1-hydroxy-2,6... Exemptions From Tolerances § 180.1281 S-Abscisic Acid, (S)-5-(1-hydroxy-2,6,6-trimethyl-4-oxo-1-cyclohex-2... from the requirement of a tolerance is established for residues of S-Abscisic Acid in or on all food...

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

PubMed

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

2012-10-01

Engineered transcription activator-like effector nucleases (TALENs) are broadly useful tools for performing targeted genome editing in a wide variety of organisms and cell types including plants, zebrafish, C. elegans, rat, human somatic cells, and human pluripotent stem cells. Here we describe detailed protocols for the serial, hierarchical assembly of TALENs that require neither PCR nor specialized multi-fragment ligations and that can be implemented by any laboratory. These restriction enzyme and ligation (REAL)-based protocols can be practiced using plasmid libraries and user-friendly, Web-based software that both identifies target sites in sequences of interest and generates printable graphical guides that facilitate assembly of TALENs. With the described platform of reagents, protocols, and software, researchers can easily engineer multiple TALENs within 2 weeks using standard cloning techniques. 2012 by John Wiley & Sons, Inc.

Avirulent Marek’s Disease Virus Type 1 Strain 814 Vectored Vaccine Expressing Avian Influenza (AI) Virus H5 Haemagglutinin Induced Better Protection Than Turkey Herpesvirus Vectored AI Vaccine

PubMed Central

Cui, Xianlan; Zhao, Yan; Shi, Xingming; Li, Qiaoling; Yan, Shuai; Gao, Ming; Wang, Mei; Liu, Changjun; Wang, Yunfeng

2013-01-01

Background Herpesvirus of turkey (HVT) as a vector to express the haemagglutinin (HA) of avian influenza virus (AIV) H5 was developed and its protection against lethal Marek’s disease virus (MDV) and highly pathogenic AIV (HPAIV) challenges was evaluated previously. It is well-known that avirulemt MDV type 1 vaccines are more effective than HVT in prevention of lethal MDV infection. To further increase protective efficacy against HPAIV and lethal MDV, a recombinant MDV type 1 strain 814 was developed to express HA gene of HPAIV H5N1. Methodology/Principal Findings A recombinant MDV-1 strain 814 expressing HA gene of HPAIV H5N1 virus A/goose/Guangdong/3/96 at the US2 site (rMDV-HA) was developed under the control of a human CMV immediate-early promoter. The HA expression in the rMDV-HA was tested by immunofluorescence and Western blot analyses, and in vitro and in vivo growth properties of rMDV-HA were also analyzed. Furthermore, we evaluated and compared the protective immunity of rMDV-HA and previously constructed rHVT-HA against HPAIV and lethal MDV. Vaccination of chickens with rMDV-HA induced 80% protection against HPAIV, which was better than the protection rate by rHVT-HA (66.7%). In the animal study with MDV challenge, chickens immunized with rMDV-HA were completely protected against virulent MDV strain J-1 whereas rHVT-HA only induced 80% protection with the same challenge dose. Conclusions/Significance The rMDV-HA vaccine was more effective than rHVT-HA vaccine for protection against lethal MDV and HPAIV challenges. Therefore, avirulent MDV type 1 vaccine is a better vector than HVT for development of a recombinant live virus vaccine against virulent MDV and HPAIV in poultry. PMID:23301062

75 FR 70937 - U.S. Customs and Border Protection

Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-19

... Robinson International (USA) Inc., as a Commercial Gauger and Laboratory AGENCY: U.S. Customs and Border Protection, Department of Homeland Security. ACTION: Notice of accreditation and approval of Robinson... to 19 CFR 151.12 and 19 CFR 151.13, Robinson International (USA) Inc., 4400 S. Wayside Drive, Suite...

Biophysical Characterization and Predicted Human Thermal Responses to U.S. Army Body Armor Protection Levels (BAPL)

DTIC Science & Technology

2013-09-01

for ballistic protection in the form of hard (e.g., ceramic plates ) and soft armor materials. The ultimate goal of these protective vests is to...Strain Decision Aid im Vapor permeability im/clo permeability index IOTV Interceptor Outer Tactical Vest m•s meters per second PC Plate Carrier RH...Army Body Armor Protection Levels (BAPL) 0 to 5 Level Configuration Added Weight lbs/kg BAPL 0 No body armor 0 BAPL 1 Vest or plate carrier with

Survivors Remorse: antibody-mediated protection against HIV-1.

PubMed

Lewis, George K; Pazgier, Marzena; DeVico, Anthony L

2017-01-01

It is clear that antibodies can play a pivotal role in preventing the transmission of HIV-1 and large efforts to identify an effective antibody-based vaccine to quell the epidemic. Shortly after HIV-1 was discovered as the cause of AIDS, the search for epitopes recognized by neutralizing antibodies became the driving strategy for an antibody-based vaccine. Neutralization escape variants were discovered shortly thereafter, and, after almost three decades of investigation, it is now known that autologous neutralizing antibody responses and their selection of neutralization resistant HIV-1 variants can lead to broadly neutralizing antibodies in some infected individuals. This observation drives an intensive effort to identify a vaccine to elicit broadly neutralizing antibodies. In contrast, there has been less systematic study of antibody specificities that must rely mainly or exclusively on other protective mechanisms, although non-human primate (NHP) studies as well as the RV144 vaccine trial indicate that non-neutralizing antibodies can contribute to protection. Here we propose a novel strategy to identify new epitope targets recognized by these antibodies for which viral escape is unlikely or impossible. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Electronic and geometric structures of Au30 clusters: a network of 2e-superatom Au cores protected by tridentate protecting motifs with u3-S

NASA Astrophysics Data System (ADS)

Tian, Zhimei; Cheng, Longjiu

2015-12-01

Density functional theory calculations have been performed to study the experimentally synthesized Au30S(SR)18 and two related Au30(SR)18 and Au30S2(SR)18 clusters. The patterns of thiolate ligands on the gold cores for the three thiolate-protected Au30 nanoclusters are on the basis of the ``divide and protect'' concept. A novel extended protecting motif with u3-S, S(Au2(SR)2)2AuSR, is discovered, which is termed the tridentate protecting motif. The Au cores of Au30S(SR)18, Au30(SR)18 and Au30S2(SR)18 clusters are Au17, Au20 and Au14, respectively. The superatom-network (SAN) model and the superatom complex (SAC) model are used to explain the chemical bonding patterns, which are verified by chemical bonding analysis based on the adaptive natural density partitioning (AdNDP) method and aromatic analysis on the basis of the nucleus-independent chemical shift (NICS) method. The Au17 core of the Au30S(SR)18 cluster can be viewed as a SAN of one Au6 superatom and four Au4 superatoms. The shape of the Au6 core is identical to that revealed in the recently synthesized Au18(SR)14 cluster. The Au20 core of the Au30(SR)18 cluster can be viewed as a SAN of two Au6 superatoms and four Au4 superatoms. The Au14 core of Au30S2(SR)18 can be regarded as a SAN of two pairs of two vertex-sharing Au4 superatoms. Meanwhile, the Au14 core is an 8e-superatom with 1S21P6 configuration. Our work may aid understanding and give new insights into the chemical synthesis of thiolate-protected Au clusters.Density functional theory calculations have been performed to study the experimentally synthesized Au30S(SR)18 and two related Au30(SR)18 and Au30S2(SR)18 clusters. The patterns of thiolate ligands on the gold cores for the three thiolate-protected Au30 nanoclusters are on the basis of the ``divide and protect'' concept. A novel extended protecting motif with u3-S, S(Au2(SR)2)2AuSR, is discovered, which is termed the tridentate protecting motif. The Au cores of Au30S(SR)18, Au30(SR)18 and Au30S

Noise pollution is pervasive in U.S. protected areas.

PubMed

Buxton, Rachel T; McKenna, Megan F; Mennitt, Daniel; Fristrup, Kurt; Crooks, Kevin; Angeloni, Lisa; Wittemyer, George

2017-05-05

Anthropogenic noise threatens ecological systems, including the cultural and biodiversity resources in protected areas. Using continental-scale sound models, we found that anthropogenic noise doubled background sound levels in 63% of U.S. protected area units and caused a 10-fold or greater increase in 21%, surpassing levels known to interfere with human visitor experience and disrupt wildlife behavior, fitness, and community composition. Elevated noise was also found in critical habitats of endangered species, with 14% experiencing a 10-fold increase in sound levels. However, protected areas with more stringent regulations had less anthropogenic noise. Our analysis indicates that noise pollution in protected areas is closely linked with transportation, development, and extractive land use, providing insight into where mitigation efforts can be most effective. Copyright © 2017, American Association for the Advancement of Science.

H2S Protects Against Methionine–Induced Oxidative Stress in Brain Endothelial Cells

PubMed Central

Tyagi, Neetu; Moshal, Karni S.; Sen, Utpal; Vacek, Thomas P.; Kumar, Munish; Hughes, William M.; Kundu, Soumi

2009-01-01

Abstract Homocysteine (Hcy) causes cerebrovascular dysfunction by inducing oxidative stress. However, to date, there are no strategies to prevent Hcy-induced oxidative damage. Hcy is an H2S precursor formed from methionine (Met) metabolism. We aimed to investigate whether H2S ameliorated Met-induced oxidative stress in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to Met treatment in the presence or absence of NaHS (donor of H2S). Met-induced cell toxicity increased the levels of free radicals in a concentration-dependent manner. Met increased NADPH-oxidase-4 (NOX-4) expression and mitigated thioredxion-1(Trx-1) expression. Pretreatment of bEnd3 with NaHS (0.05 mM) attenuated the production of free radicals in the presence of Met and protected the cells from oxidative damage. Furthermore, NaHS enhanced inhibitory effects of apocynin, N-acetyl-l-cysteine (NAC), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), Nω-nitro-l-arginine methyl ester (L-NAME) on ROS production and redox enzymes levels induced by Met. In conclusion, the administration of H2S protected the cells from oxidative stress induced by hyperhomocysteinemia (HHcy), which suggested that NaHS/H2S may have therapeutic potential against Met-induced oxidative stress. Antioxid. Redox Signal. 11, 25–33. PMID:18837652

Site-Specific Editing of the Plasmodium falciparum Genome Using Engineered Zinc-Finger Nucleases

PubMed Central

Straimer, Judith; Lee, Marcus CS; Lee, Andrew H; Zeitler, Bryan; Williams, April E; Pearl, Jocelynn R; Zhang, Lei; Rebar, Edward J; Gregory, Philip D; Llinás, Manuel; Urnov, Fyodor D; Fidock, David A

2013-01-01

Malaria afflicts over 200 million people worldwide and its most lethal etiologic agent, Plasmodium falciparum, is evolving to resist even the latest-generation therapeutics. Efficient tools for genome-directed investigations of P. falciparum pathogenesis, including drug resistance mechanisms, are clearly required. Here we report rapid and targeted genetic engineering of this parasite, using zinc-finger nucleases (ZFNs) that produce a double-strand break in a user-defined locus and trigger homology-directed repair. Targeting an integrated egfp locus, we obtained gene deletion parasites with unprecedented speed (two weeks), both with and without direct selection. ZFNs engineered against the endogenous parasite gene pfcrt, responsible for chloroquine treatment escape, rapidly produced parasites that carried either an allelic replacement or a panel of specified point mutations. The efficiency, versatility and precision of this method will enable a diverse array of genome editing approaches to interrogate this human pathogen. PMID:22922501

PD-1 suppresses protective immunity to Streptococcus pneumoniae through a B cell-intrinsic mechanism

PubMed Central

McKay, Jerome T.; Egan, Ryan P.; Yammani, Rama D.; Chen, Lieping; Shin, Tahiro; Yagita, Hideo; Haas, Karen M.

2015-01-01

Despite the emergence of the PD-1:PD-1 ligand (PD-L) regulatory axis as a promising target for treating multiple human diseases, remarkably little is known about how this pathway regulates responses to extracellular bacterial infections. We found that PD-1−/− mice, as well as wild type mice treated with a PD-1 blocking antibody, exhibited significantly increased survival against lethal Streptococcus pneumoniae infection following either priming with low-dose pneumococcal respiratory infection or S. pneumoniae-capsular polysaccharide immunization. Enhanced survival in mice with disrupted PD-1:PD-L interactions was explained by significantly increased proliferation, isotype switching, and IgG production by pneumococcal capsule-specific B cells. Both PD-1 ligands, B7-H1 and B7-DC, contributed to PD-1-mediated suppression of protective capsule-specific IgG. Importantly, PD-1 was induced on capsule-specific B cells and suppressed IgG production and protection against pneumococcal infection in a B cell-intrinsic manner. These results provide the first demonstration of a physiologic role for B cell-intrinsic PD-1 expression in vivo. In summary, our study reveals that B cell-expressed PD-1 plays a central role in regulating protection against S. pneumoniae, and thereby represents a promising target for bolstering immunity to encapsulated bacteria. PMID:25624454

43 CFR 2361.1 - Protection of the environment.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 43 Public Lands: Interior 2 2011-10-01 2011-10-01 false Protection of the environment. 2361.1... Management and Protection of the National Petroleum Reserve in Alaska § 2361.1 Protection of the environment... the environment during such activities in the Reserve, and other related activities. (c) Maximum...

43 CFR 2361.1 - Protection of the environment.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 43 Public Lands: Interior 2 2014-10-01 2014-10-01 false Protection of the environment. 2361.1... Management and Protection of the National Petroleum Reserve in Alaska § 2361.1 Protection of the environment... the environment during such activities in the Reserve, and other related activities. (c) Maximum...

43 CFR 2361.1 - Protection of the environment.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 43 Public Lands: Interior 2 2013-10-01 2013-10-01 false Protection of the environment. 2361.1... Management and Protection of the National Petroleum Reserve in Alaska § 2361.1 Protection of the environment... the environment during such activities in the Reserve, and other related activities. (c) Maximum...

43 CFR 2361.1 - Protection of the environment.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 43 Public Lands: Interior 2 2012-10-01 2012-10-01 false Protection of the environment. 2361.1... Management and Protection of the National Petroleum Reserve in Alaska § 2361.1 Protection of the environment... the environment during such activities in the Reserve, and other related activities. (c) Maximum...

Chimeric severe acute respiratory syndrome coronavirus (SARS-CoV) S glycoprotein and influenza matrix 1 efficiently form virus-like particles (VLPs) that protect mice against challenge with SARS-CoV

PubMed Central

Liu, Ye V.; Massare, Michael J.; Barnard, Dale L.; Kort, Thomas; Nathan, Margret; Wang, Lei; Smith, Gale

2011-01-01

SARS-CoV was the cause of the global pandemic in 2003 that infected over 8000 people in 8 months. Vaccines against SARS are still not available. We developed a novel method to produce high levels of a recombinant SARS virus-like particles (VLPs) vaccine containing the SARS spike (S) protein and the influenza M1 protein using the baculovirus insect cell expression system. These chimeric SARS VLPs have a similar size and morphology to the wild type SARS-CoV. We tested the immunogenicity and protective efficacy of purified chimeric SARS VLPs and full length SARS S protein vaccines in a mouse lethal challenge model. The SARS VLP vaccine, containing 0.8 μg of SARS S protein, completely protected mice from death when administered intramuscular (IM) or intranasal (IN) routes in the absence of an adjuvant. Likewise, the SARS VLP vaccine, containing 4 μg of S protein without adjuvant, reduced lung virus titer to below detectable level, protected mice from weight loss, and elicited a high level of neutralizing antibodies against SARS-CoV. Sf9 cell-produced full length purified SARS S protein was also an effective vaccine against SARS-CoV but only when co-administered IM with aluminum hydroxide. SARS-CoV VLPs are highly immunogenic and induce neutralizing antibodies and provide protection against lethal challenge. Sf9 cell-based VLP vaccines are a potential tool to provide protection against novel pandemic agents. PMID:21762752

U.S. ENVIRONMENTAL PROTECTION AGENCY'S REGULATION AND MANAGEMENT OF WATERBORNE VIRUSES

EPA Science Inventory

The U.S. Environmental Protection Agency (USEPA) manages waterborne viruses and other pathogens through the establishment of rules and regulations that are designed to ensure public health protection. The rules that currently regulate pathogens focus on the management of viruses...

Histone deacetylase inhibition rescues gene knockout levels achieved with integrase-defective lentiviral vectors encoding zinc-finger nucleases.

PubMed

Pelascini, Laetitia P L; Maggio, Ignazio; Liu, Jin; Holkers, Maarten; Cathomen, Toni; Gonçalves, Manuel A F V

2013-12-01

Zinc-finger nucleases (ZFNs) work as dimers to induce double-stranded DNA breaks (DSBs) at predefined chromosomal positions. In doing so, they constitute powerful triggers to edit and to interrogate the function of genomic sequences in higher eukaryotes. A preferred route to introduce ZFNs into somatic cells relies on their cotransduction with two integrase-defective lentiviral vectors (IDLVs) each encoding a monomer of a functional heterodimeric pair. The episomal nature of IDLVs diminishes the risk of genotoxicity and ensures the strict transient expression profile necessary to minimize deleterious effects associated with long-term ZFN activity. However, by deploying IDLVs and conventional lentiviral vectors encoding HPRT1- or eGFP-specific ZFNs, we report that DSB formation at target alleles is limited after IDLV-mediated ZFN transfer. This IDLV-specific underperformance stems, to a great extent, from the activity of chromatin-remodeling histone deacetylases (HDACs). Importantly, the prototypic and U.S. Food and Drug Administration-approved inhibitors of metal-dependent HDACs, trichostatin A and vorinostat, respectively, did not hinder illegitimate recombination-mediated repair of targeted chromosomal DSBs. This allowed rescuing IDLV-mediated site-directed mutagenesis to levels approaching those achieved by using their isogenic chromosomally integrating counterparts. Hence, HDAC inhibition constitutes an efficacious expedient to incorporate in genome-editing strategies based on transient IDLV-mediated ZFN expression. Finally, we compared two of the most commonly used readout systems to measure targeted gene knockout activities based on restriction and mismatch-sensitive endonucleases. These experiments indicate that these enzymatic assays display a similar performance.

Environmental Public Health Research at the U.S. Environmental Protection Agency – Addressing Community Exposures and Outcomes from One Researcher’s Perspective

EPA Science Inventory

The U.S. Environmental Protection Agency’s (U.S. EPA) mission is to protect human health and the environment. Those not familiar with U.S. EPA’s mission often do not realize that U.S. EPA is a public health agency. In this presentation, Dr. Danelle Lobdell will provid...

Assessing Cumulative Impact and Risk - Approaches at the U.S. Environmental Protection Agency

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) has a mission and regulatory mandate to protect human health and the environment. EPA’s primary role is to implement environmental laws by developing and enforcing national regulation. Cogent to the goals of this workshop, key envi...

Bite protection analysis of permethrin-treated U.S. Military uniforms

USDA-ARS?s Scientific Manuscript database

Historically, combat casualties from diseases have greatly outnumbered battle injuries received from actual combat during military operations. Since 1951, United States military combat uniforms have been treated within insecticides to protect personnel from arthropod attack. In the 1970s and 1980s,...

Synthesis and in vitro characterization of a novel S-protected thiolated alginate.

PubMed

Hauptstein, Sabine; Dezorzi, Stefanie; Prüfert, Felix; Matuszczak, Barbara; Bernkop-Schnürch, Andreas

2015-06-25

The object of this study was to synthesize and characterize a novel S-protected thiolated polymer with a high degree of modification. In this regard, an alginate-cysteine and an alginate-cysteine-2-mercaptonicotinic acid conjugate were synthesized. To achieve a high coupling rate of the thiol group bearing ligand cysteine to the polymer, the carbohydrate was activated by an oxidative ring opening with sodium periodate followed by a reductive amination to bind the primary amino group of cysteine to resulting reactive aldehyde groups. The obtained thiolated polymer displayed 1561±130μmol thiol groups per gram polymer. About one third of these thiol groups were S-protected by the implementation of a thiol bearing aromatic protection group via disulfide bond formation. Test tablets of both modified polymers showed improved stability against oxidation in aqueous environment compared to the unmodified alginate and exhibit higher water-uptake capacity. Rheological investigations revealed an increased viscosity of the S-protected thiolated polymer whereat the thiolated non S-protected polymer showed gelling properties after the addition of hydrogen peroxide. The mucoadhesive properties could be improved significantly for both derivatives and no alteration in biocompatibility tested on Caco-2 cell monolayer employing an MTT assay could be detected after modification. According to these results, both new derivatives seem promising for various applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

U.S. ENVIRONMENTAL PROTECTION AGENCY'S COMPUTATIONAL TOXCIOLOGY PROGRAM - METABOLISM AND METABONOMICS

EPA Science Inventory

The mission of the U. S. Environmental Protection Agency (EPA) is to safeguard public health and the environment from adverse effects that may be caused by exposure to pollutants in the air, water, soil and food. Protecting human health and the environment carries with it the ch...

High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

PubMed

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

PubMed Central

Gao, Dandan; Ding, Chen; Li, Zhihua; Li, Yadong; Cun, Wei; Li, Qihan

2014-01-01

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. PMID:24788700

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rashid, Fahad; Harris, Paul D.; Zaher, Manal S.

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 missesmore » 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.« less

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

DOE PAGES

Rashid, Fahad; Harris, Paul D.; Zaher, Manal S.; ...

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 missesmore » 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.« less

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

PubMed Central

Rashid, Fahad; Harris, Paul D; Zaher, Manal S; Sobhy, Mohamed A; Joudeh, Luay I; Yan, Chunli; Piwonski, Hubert; Tsutakawa, Susan E; Ivanov, Ivaylo; Tainer, John A; Habuchi, Satoshi; Hamdan, Samir M

2017-01-01

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. DOI: http://dx.doi.org/10.7554/eLife.21884.001 PMID:28230529

37 CFR 7.28 - Replacement of U.S. registration by registered extension of protection.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false Replacement of U.S. registration by registered extension of protection. 7.28 Section 7.28 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE, DEPARTMENT OF COMMERCE RULES OF PRACTICE IN FILINGS PURSUANT...

Systems analysis of protective immune responses to RTS,S malaria vaccination in humans

PubMed Central

Kazmin, Dmitri; Nakaya, Helder I.; Lee, Eva K.; Johnson, Matthew J.; van der Most, Robbert; van den Berg, Robert A.; Ballou, W. Ripley; Jongert, Erik; Wille-Reece, Ulrike; Ockenhouse, Christian; Aderem, Alan; Zak, Daniel E.; Sadoff, Jerald; Hendriks, Jenny; Wrammert, Jens; Ahmed, Rafi; Pulendran, Bali

2017-01-01

RTS,S is an advanced malaria vaccine candidate and confers significant protection against Plasmodium falciparum infection in humans. Little is known about the molecular mechanisms driving vaccine immunity. Here, we applied a systems biology approach to study immune responses in subjects receiving three consecutive immunizations with RTS,S (RRR), or in those receiving two immunizations of RTS,S/AS01 following a primary immunization with adenovirus 35 (Ad35) (ARR) vector expressing circumsporozoite protein. Subsequent controlled human malaria challenge (CHMI) of the vaccinees with Plasmodium-infected mosquitoes, 3 wk after the final immunization, resulted in ∼50% protection in both groups of vaccinees. Circumsporozoite protein (CSP)-specific antibody titers, prechallenge, were associated with protection in the RRR group. In contrast, ARR-induced lower antibody responses, and protection was associated with polyfunctional CD4+ T-cell responses 2 wk after priming with Ad35. Molecular signatures of B and plasma cells detected in PBMCs were highly correlated with antibody titers prechallenge and protection in the RRR cohort. In contrast, early signatures of innate immunity and dendritic cell activation were highly associated with protection in the ARR cohort. For both vaccine regimens, natural killer (NK) cell signatures negatively correlated with and predicted protection. These results suggest that protective immunity against P. falciparum can be achieved via multiple mechanisms and highlight the utility of systems approaches in defining molecular correlates of protection to vaccination. PMID:28193898

Comparison of the protective effectiveness of NPQ in Arabidopsis plants deficient in PsbS protein and zeaxanthin

PubMed Central

Ware, Maxwell A.; Belgio, Erica; Ruban, Alexander V.

2015-01-01

The efficiency of protective energy dissipation by non-photochemical quenching (NPQ) in photosystem II (PSII) has been recently quantified by a new non-invasive photochemical quenching parameter, qPd. PSII yield (ФPSII) was expressed in terms of NPQ, and the extent of damage to the reaction centres (RCIIs) was calculated via qPd as: ФPSII=qPd×(F v/F m)/{1+[1–(F v/F m)]×NPQ}. Here this approach was used to determine the amount of NPQ required to protect all PSII reaction centres (pNPQ) under a gradually increasing light intensity, in the zeaxanthin-deficient (npq1) Arabidopsis mutant, compared with PsbS protein-deficient (npq4) and wild-type plants. The relationship between maximum pNPQ and tolerated light intensity for all plant genotypes followed similar trends. These results suggest that under a gradually increasing light intensity, where pNPQ is allowed to develop, it is only the amplitude of pNPQ which is the determining factor for protection. However, the use of a sudden constant high light exposure routine revealed that the presence of PsbS, not zeaxanthin, offered better protection for PSII. This was attributed to a slower development of pNPQ in plants lacking PsbS in comparison with plants that lacked zeaxanthin. This research adds further support to the value of pNPQ and qPd as effective parameters for assessing NPQ effectiveness in different types of plants. PMID:25429003

[Protective Effect of S-isopentenyl-L-cysteine against DNA Damage in Irradiated Mice].

PubMed

Zheng, Qi-sheng; Yu, Guang-yun; He, Xin; Jiang, Ming; Chu, Xiao-fei; Zhao, Shu-yi; Fan, Sai-jun; Liu, Pei-xun

2015-10-01

To evaluate the protective effect of S-isopentenyl-L-cysteine,a new cysteine derivative,on DNA damage induced by radiation by using acute radiation injury animal models. Forty ICR mice were randomly divided into five groups:the control group,1.0Gy gamma irradiation group,1.0Gy gamma irradiation combined with S-isopentenyl-L-cysteine group,7.2Gy gamma irradiation group,and 7.2Gy gamma irradiation combined with S-isopentenyl-L-cysteine group,with 8 mice in each group.The comet assay and bone marrow polychromatic micronucleus experiments were performed to evaluate the double-strand DNA breaks in ICR mice exposed to 1.0 and 7.2Gy gamma-ray, respectively. The tail DNA percentage,tail length,tail moment,and olive tail moment of peripheral blood lymphocytes in 7.2Gy gamma irradiation group were significantly higher than that of the control group (P<0.01).And it was also observed that above experimental indexes of 7.2Gy gamma irradiation combined with S-isopentenyl-L-cysteine group was significantly less than that of 7.2Gy gamma irradiation group (P<0.05). In addition,the micronucleus rate of 1.0Gy gamma irradiation group and 7.2Gy gamma irradiation group were both significantly higher than in the control group (P<0.01). In addition,in mice given S-isopentenyl-L-cysteine before irradiation,the micronucleus rate of ICR mice exposed to 1.0 and 7.2Gy gamma-ray decreased from (39.5000 ± 3.3141)‰ to (28.1667±4.1345)‰ (P=0.033) and from (76.5000 ± 4.6242)‰ to (22.8333 ± 3.6553)‰(P=0.000),respectively. The bone marrow polychromatic micronucleus experiment indicated that the value of polychromatic erythrocyte (PCE)/normochromatic erythrocyte(NCE) of ICR mice exposed to 1.0 and 7.2Gy gamma-ray was less than the control group(P<0.05). Meanwhile,after irradiating by certain dose,the value of PCE/NCE in mice given S-isopentenyl-L-cysteine before irradiation was significantly higher than the corresponding groups (P<0.05). S-isopentenyl-L-cysteine has a good protective

EPA’s proposed Worker Protection Standard and the burdens of the past

PubMed Central

Bohme, Susanna Rankin

2015-01-01

Background: An Environmental Protection Agency (EPA) recommendation for extensive changes to the Agency’s 40-year-old Worker Protection Standard is currently stalled in the “proposed rule” stage. The proposal, which was available for public comment until 18 August, would improve safety, training, and hazard communication policies for agricultural pesticides. Exposure to hazards, including high heat, heavy machinery, stoop labor, and pesticides, makes occupational illness uncommonly common among the USA’s estimated 2.5 million farm workers. Objectives: To consider the proposed revisions’ likelihood of addressing historical gaps in farmworker protection. Methods: The proposal was compared to the existing Worker Protection Standard, and key aspects were analyzed in relation to existing science on farm labor hazards, as well as historic occupational health, labor and immigration policy. Results: US law historically has left farm workers largely unprotected. These exclusions and delays have been tolerated in part thanks to the myth of the independent family farmer, but more significant is the stingy nativism that presumes to benefit from immigrant labor without assuming any responsibility to protect the humans who provide it. In the first half of the 1970s, workers lobbied for robust protections, but rule making was impeded by lack of data and by the disproportionate influence of agricultural employers who sought minimal regulation. In 1974, the EPA passed the first Worker Protection Standard for farm workers. Key aspects of the proposed revision include stronger protections against drift and re-entry exposures, better information provision and training, and increased protections for workers under 16 years. Conclusions: The proposed changes represent an improvement over existing legislation, but do not go far enough. The revision should be strengthened along lines suggested by farm workers themselves, and other labor laws must also be amended to give the men

Report: Assessing EPA’s Efforts to Protect Sensitive Information

EPA Pesticide Factsheets

Report #2006-S-00006, September 19, 2006. This is the EPA OIG's completed Data Collection Instrument, as prescribed by the PCIE to use in meeting its requirements under OMB Memo M-06-16, Protection of Sensitive Agency Information.

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

PubMed

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

2013-07-01

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

Engineering Customized TALE Nucleases (TALENs) and TALE Transcription Factors by Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) Assembly

PubMed Central

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

2013-01-01

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 multiple different 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 down-regulate expression of endogenous genes in human cells and plants. Here we describe a detailed protocol for practicing 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 multi-channel pipet. With the automated version of FLASH, a single researcher can construct up to 96 DNA fragments encoding various length TALE repeat arrays in one day and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in one week or less. Plas-mids required to practice FLASH are available by request from the Joung Lab (http://www.jounglab.org/). We also describe here improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) webserver (http://ZiFiTBeta.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high-throughput. PMID:23821439

Use of protective eyewear in U.S. adults: results from the 2002 national health interview survey.

PubMed

Forrest, Kimberly Y Z; Cali, Joseph M; Cavill, Wilma J

2008-01-01

Many eye injuries occur because of not using protective eyewear. This study analyzed the 2002 National Health Interview Survey data to examine the rate of using protective eyewear during activities that could cause eye injuries outside the workplace and the correlates of not using protective eyewear among U.S. adults. The rate of participation in activities that could cause eye injuries and the rate of protective eyewear use during these activities were estimated using sample weights to yield national estimates. A total of 30,894 individuals in the survey provided valid data for the current analysis, which revealed that 29.3% of the U.S. adults reported engaging in activities that could cause an eye injury and 32.1% of those used eye protection while doing such activities. Males were more likely to use eye protection than females (34.7% vs. 25.2%). The age group of 18-24 years was least likely to use eye protection (15.3%). Other factors associated with not using protective eyewear included being black or other races, non-retired individuals, and those from low-income families (all p-values < 0.05). Use of protective eyewear during activities that could cause eye injuries was relatively low in the US adult population. Certain groups were at a higher risk for not using eye protection, including females, young adults aged 18-24 years, black or other races, and individuals with a low socioeconomic status. To promote eye safety, health education programs should target these high-risk groups.

A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease.

PubMed

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.

An ion-conductive Li1.5Al0.5Ge1.5(PO4)3-based composite protective layer for lithium metal anode in lithium-sulfur batteries

NASA Astrophysics Data System (ADS)

Sun, Changzhi; Huang, Xiao; Jin, Jun; Lu, Yang; Wang, Qing; Yang, Jianhua; Wen, Zhaoyin

2018-02-01

Lithium (Li) metal plays an indispensable role in Li-S batteries, but its fast degradation seriously impedes the practical application of Li-S batteries. Here, an ion-conductive LAGP-based composite protective layer (CPL) consisting of lithium aluminum germanium phosphate (Li1.5Al0.5Ge1.5(PO4)3) and polyvinylidene fluoride (PVDF) is prepared on Li metal anode via a facile casting method. In the presence of LAGP, the negative effect of CPL on the diffusion of Li+ is minimized. Hence, it can not only effectively resist corrosive action of lithium polysulfides (LiPSs) on Li metal anode, but also reduce interfacial polarization and restrain dendritic Li growth. The protected Li anode exhibits exceptional cycling stability and low voltage polarization (∼30 mV at 0.5 mA cm-2) for 300 h. The electrochemical performance of Li-S batteries with protected Li anode is also greatly enhanced. The discharge specific capacity of 832.1 mAh g-1 and an average coulombic efficiency of 92% are obtained for up to 100 cycles at 0.5 C in LiNO3-free electrolyte. Additionally, the rate capability of Li-S batteries is significantly improved, delivering a reversible capacity of 565 mAh g-1 at 4 C. Our results also indicate this protection strategy can be extended to the Li-S pouch cells.

Gene repair of an Usher syndrome causing mutation by zinc-finger nuclease mediated homologous recombination.

PubMed

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.

Sirt1 Protects Stressed Adult Hematopoietic Stem Cells | Center for Cancer Research

Cancer.gov

The immune system relies on a stable pool of hematopoietic stem and progenitor cells (HSPCs) to respond properly to injury or stress. Maintaining genomic integrity and appropriate gene expression is essential for HSPC homeostasis, and dysregulation can result in myeloproliferative disorders or loss of immune function. Sirt1 is a histone deacetylase that can protect embryonic stem (ES) cells from accumulating DNA damage and has been linked to hematopoietic differentiation of ES cells. Satyendra Singh, Ph.D., a postdoctoral fellow working with Philipp Oberdoerffer, Ph.D., in CCR’s Laboratory of Receptor Biology and Gene Expression, and their colleagues set out to determine whether Sirt1 could play a similar protective role in adult HSPCs.

The nucleotide sequence of the intergenic region between the 5.8S and 26S rRNA genes of the yeast ribosomal RNA operon. Possible implications for the interaction between 5.8S and 26S rRNA and the processing of the primary transcript.

PubMed Central

Veldman, G M; Klootwijk, J; van Heerikhuizen, H; Planta, R J

1981-01-01

We have determined the nucleotide sequence of part of a cloned yeast ribosomal RNA operon extending from the 5.8S RNA gene downstream into the 5' -terminal region of the 26S RNA gene. We mapped the pertinent processing sites, viz. the 5' end of 26S rRNA and the 3'ends of 5.8S rRNA and its immediate precursor, 7S RNA. At the 3' end of 7S RNA we find the sequence UCGUUU which is very similar to the type I consensus sequence UCAUUA/U present at the 3' ends of 17S, 5.8S and 26S rRNA as well as 18S precursor rRNA in yeast. At the 5' end of the 26S RNA gene we find a sequence of thirteen nucleotides which is homologous to the type II sequence present at the 5' termini of both the 17S and the 5.8S RNA gene. These findings further support the suggestion put forward earlier (G.M. Veldman et al. (1980) Nucl. Acids Res. 8, 2907-2920) that both consensus sequences are involved in the recognition of precursor rRNA by the processing nuclease(s). We discuss a model for the processing of yeast rRNA in which a processing enzyme sequentially recognizes several combinations of a type I and a type II consensus sequence. We also describe the existence of a significant base complementarity between sequences in the 5' -terminal region of 26S rRNA and the 3' -terminal region of 5.8S rRNA. We suggest that base pairing between these sequences contributes to the binding between 5.8S and 26S rRNA. Images PMID:7312619

The axon-protective WLD(S) protein partially rescues mitochondrial respiration and glycolysis after axonal injury.

PubMed

Godzik, Katharina; Coleman, Michael P

2015-04-01

The axon-protective Wallerian degeneration slow (WLD(S)) protein can ameliorate the decline in axonal ATP levels after neurite transection. Here, we tested the hypothesis that this effect is associated with maintenance of mitochondrial respiration and/or glycolysis. We used isolated neurites of superior cervical ganglion (SCG) cultures in the Seahorse XF-24 Metabolic Flux Analyser to determine mitochondrial respiration and glycolysis under different conditions. We observed that both mitochondrial respiration and glycolysis declined significantly during the latent phase of Wallerian degeneration. WLD(S) partially reduced the decline both in glycolysis and in mitochondrial respiration. In addition, we found that depleting NAD levels in uncut cultures led to changes in mitochondrial respiration and glycolysis similar to those rescued by WLD(S) after cut, suggesting that the maintenance of NAD levels in Wld(S) neurites after axonal injury at least partially underlies the maintenance of ATP levels. However, by using another axon-protective mutation (Sarm1(-/-)), we could demonstrate that rescue of basal ECAR (and hence probably glycolysis) rather than basal OCR (mitochondrial respiration) may be part of the protective phenotype to delay Wallerian degeneration. These findings open new routes to study glycolysis and the connection between NAD and ATP levels in axon degeneration, which may help to eventually develop therapeutic strategies to treat neurodegenerative diseases.

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

PubMed

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.

Immunization to Protect the U.S. Armed Forces: Heritage, Current Practice, Prospects

DTIC Science & Technology

2005-01-01

population . (4, 12, 16, 163, 164) This vaccine was derived from virus- infected mouse brain. Immunization to Protect the U.S. Armed Forces 14...individual health and to keep units strong so they can accomplish their military missions. The FDA-licensed vaccines selected protect against infections ...CA. Passive protection of mice against lethal Francisella tularensis (live tularemia vaccine strain) infection by the sera of human recipients of

Environmental Protection Versus Intellectual Property: The U.S-Mexico Free Trade Agreement Negotiations.

ERIC Educational Resources Information Center

Foy, George

1992-01-01

Compares the U.S. objective of protecting the knowledge required to develop certain products to environmental protection in the United States-Mexico Free Trade Act negotiations. Argues that environmental regulation should be included in the negotiations with force equal to that for intellectual rights protection. (55 references) (MDH)

IGF-1 Protects Dopamine Neurons Against Oxidative Stress: Association with Changes in Phosphokinases

PubMed Central

El Ayadi, Amina; Zigmond, Michael J.; Smith, Amanda D.

2016-01-01

Insulin-like growth factor-1 (IGF-1) is an endogenous peptide transported across the blood brain barrier that is protective in several brain injury models, including an acute animal model of Parkinson’s disease (PD). Motor deficits in PD are due largely to the progressive loss of nigrostriatal dopaminergic neurons. Thus, we examined the neuroprotective potential of IGF-1 in a progressive model of dopamine deficiency in which 6-hydroxydopamine (6-OHDA) is infused into the striatum. Rats received intrastriatal IGF-1 (5 or 50 μg) 6 hrs prior to infusion of 4 μg 6-OHDA into the same site and were sacrificed 1 or 4 wks later. Both concentrations of IGF-1 protected tyrosine hydroxylase (TH) immunoreactive terminals in striatum at 4 wks but not at 1 wk, indicating that IGF-induced restoration of the dopaminergic phenotype occurred over several weeks. TH-immunoreactive cell loss was only attenuated with 50 μg IGF-1. We then examined the effect of striatal IGF-1 on the Ras/ERK1/2 and PI3K/Akt pathways to ascertain if their activation correlated with IGF-1-induced protection. Striatal and nigral levels of phospho-ERK1/2 (pERK1/2) were maximal 6 hrs after IGF-1 infusion and, with the exception of an increase in nigral pERK2 at 48 hrs, returned to basal levels by 7 days. Phospho-Akt (Ser473) was elevated 6–24 hrs post-IGF-1 infusion in both striatum and substantia nigra concomitant with inhibition of pro-death GSK-3β, a downstream target of Akt. These results suggest that IGF-1 can protect the nigrostriatal pathway in a progressive PD model and that this protection is preceded by activation of key pro-survival signaling cascades PMID:26894890

Comparison of the protective effectiveness of NPQ in Arabidopsis plants deficient in PsbS protein and zeaxanthin.

PubMed

Ware, Maxwell A; Belgio, Erica; Ruban, Alexander V

2015-03-01

The efficiency of protective energy dissipation by non-photochemical quenching (NPQ) in photosystem II (PSII) has been recently quantified by a new non-invasive photochemical quenching parameter, qPd. PSII yield (ФPSII) was expressed in terms of NPQ, and the extent of damage to the reaction centres (RCIIs) was calculated via qPd as: ФPSII=qPd×(F v/F m)/{1+[1-(F v/F m)]×NPQ}. Here this approach was used to determine the amount of NPQ required to protect all PSII reaction centres (pNPQ) under a gradually increasing light intensity, in the zeaxanthin-deficient (npq1) Arabidopsis mutant, compared with PsbS protein-deficient (npq4) and wild-type plants. The relationship between maximum pNPQ and tolerated light intensity for all plant genotypes followed similar trends. These results suggest that under a gradually increasing light intensity, where pNPQ is allowed to develop, it is only the amplitude of pNPQ which is the determining factor for protection. However, the use of a sudden constant high light exposure routine revealed that the presence of PsbS, not zeaxanthin, offered better protection for PSII. This was attributed to a slower development of pNPQ in plants lacking PsbS in comparison with plants that lacked zeaxanthin. This research adds further support to the value of pNPQ and qPd as effective parameters for assessing NPQ effectiveness in different types of plants. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Protective immunity to Japanese encephalitis virus associated with anti-NS1 antibodies in a mouse model.

PubMed

Li, Yize; Counor, Dorian; Lu, Peng; Duong, Veasna; Yu, Yongxin; Deubel, Vincent

2012-07-24

Japanese encephalitis virus (JEV) is a major mosquito-borne pathogen that causes viral encephalitis throughout Asia. Vaccination with an inactive JEV particle or attenuated virus is an efficient preventative measure for controlling infection. Flavivirus NS1 protein is a glycoprotein secreted during viral replication that plays multiple roles in the viral life cycle and pathogenesis. Utilizing JEV NS1 as an antigen in viral vectors induces a limited protective immune response against infection. Previous studies using E. coli-expressed JEV NS1 to immunize mice induced protection against lethal challenge; however, the protection mechanism through cellular and humoral immune responses was not described. JEV NS1 was expressed in and purified from Drosophila S2 cells in a native glycosylated multimeric form, which induced T-cell and antibody responses in immunized C3H/HeN mice. Mice vaccinated with 1 μg NS1 with or without water-in-oil adjuvant were partially protected against viral challenge and higher protection was observed in mice with higher antibody titers. IgG1 was preferentially elicited by an adjuvanted NS1 protein, whereas a larger load of IFN-γ was produced in splenocytes from mice immunized with aqueous NS1. Mice that passively received anti-NS1 mouse polyclonal immune sera were protected, and this phenomenon was dose-dependent, whereas protection was low or delayed after the passive transfer of anti-NS1 MAbs. The purified NS1 subunit induced protective immunity in relation with anti-NS1 IgG1 antibodies. NS1 protein efficiently stimulated Th1-cell proliferation and IFN-γ production. Protection against lethal challenge was elicited by passive transfer of anti-NS1 antisera, suggesting that anti-NS1 antibodies play a substantial role in anti-viral immunity.

Pressure-jump small-angle x-ray scattering detected kinetics of staphylococcal nuclease folding.

PubMed Central

Woenckhaus, J; Köhling, R; Thiyagarajan, P; Littrell, K C; Seifert, S; Royer, C A; Winter, R

2001-01-01

The kinetics of chain disruption and collapse of staphylococcal nuclease after positive or negative pressure jumps was monitored by real-time small-angle x-ray scattering under pressure. We used this method to probe the overall conformation of the protein by measuring its radius of gyration and pair-distance-distribution function p(r) which are sensitive to the spatial extent and shape of the particle. At all pressures and temperatures tested, the relaxation profiles were well described by a single exponential function. No fast collapse was observed, indicating that the rate limiting step for chain collapse is the same as that for secondary and tertiary structure formation. Whereas refolding at low pressures occurred in a few seconds, at high pressures the relaxation was quite slow, approximately 1 h, due to a large positive activation volume for the rate-limiting step for chain collapse. A large increase in the system volume upon folding implies significant dehydration of the transition state and a high degree of similarity in terms of the packing density between the native and transition states in this system. This study of the time-dependence of the tertiary structure in pressure-induced folding/unfolding reactions demonstrates that novel information about the nature of protein folding transitions and transition states can be obtained from a combination of small-angle x-ray scattering using high intensity synchrotron radiation with the high pressure perturbation technique. PMID:11222312

Novel Catanionic Surfactant Vesicle Vaccines Protect against Francisella tularensis LVS and Confer Significant Partial Protection against F. tularensis Schu S4 Strain

PubMed Central

Richard, Katharina; Mann, Barbara J.; Stocker, Lenea; Barry, Eileen M.; Qin, Aiping; Cole, Leah E.; Hurley, Matthew T.; Ernst, Robert K.; Michalek, Suzanne M.; Stein, Daniel C.; DeShong, Philip

2014-01-01

Francisella tularensis is a Gram-negative immune-evasive coccobacillus that causes tularemia in humans and animals. A safe and efficacious vaccine that is protective against multiple F. tularensis strains has yet to be developed. In this study, we tested a novel vaccine approach using artificial pathogens, synthetic nanoparticles made from catanionic surfactant vesicles that are functionalized by the incorporation of either F. tularensis type B live vaccine strain (F. tularensis LVS [LVS-V]) or F. tularensis type A Schu S4 strain (F. tularensis Schu S4 [Schu S4-V]) components. The immunization of C57BL/6 mice with “bare” vesicles, which did not express F. tularensis components, partially protected against F. tularensis LVS, presumably through activation of the innate immune response, and yet it failed to protect against the F. tularensis Schu S4 strain. In contrast, immunization with LVS-V fully protected mice against intraperitoneal (i.p.) F. tularensis LVS challenge, while immunization of mice with either LVS-V or Schu S4-V partially protected C57BL/6 mice against an intranasal (i.n.) F. tularensis Schu S4 challenge and significantly increased the mean time to death for nonsurvivors, particularly following the i.n. and heterologous (i.e., i.p./i.n.) routes of immunization. LVS-V immunization, but not immunization with empty vesicles, elicited high levels of IgG against nonlipopolysaccharide (non-LPS) epitopes that were increased after F. tularensis LVS challenge and significantly increased early cytokine production. Antisera from LVS-V-immunized mice conferred passive protection against challenge with F. tularensis LVS. Together, these data indicate that functionalized catanionic surfactant vesicles represent an important and novel tool for the development of a safe and effective F. tularensis subunit vaccine and may be applicable for use with other pathogens. PMID:24351755

Reassessment of the NRC`s program for protecting allegers against retaliation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1994-01-01

On July 6, 1993, the Nuclear Regulatory Commission`s (NRC`s) Executive Director for Operations established a review team to reassess the NRC`s program for protecting allegers against retaliation. The team evaluated the current system, and solicited comments from various NRC offices, other Federal agencies, licensees, former allegers, and the public. This report is subject to agency review. The report summarizes current processes and gives an overview of current problems. It discusses: (1) ways in which licensees can promote a quality-conscious work environment, in which all employees feel free to raise concerns without fear of retaliation; (2) ways to improve the NRC`smore » overall handling of allegations; (3) the NRC`s involvement in the Department of Labor process; (4) related NRC enforcement practices; and (5) methods other than investigation and enforcement that may be useful in treating allegations of potential or actual discrimination. Recommendations are given in each area.« less

Environmental futures research at the U.S. Environmental Protection Agency

Treesearch

Robert L. Olson

2012-01-01

Relatively little research on environmental futures has been carried out in the United States. An exception is the long-running futures research that the U.S. Environmental Protection Agency (EPA) has been conducting since the 1970s. This paper reviews past and current efforts toward developing a capacity for environmental foresight within the EPA, and discusses some...

OutKnocker: a web tool for rapid and simple genotyping of designer nuclease edited cell lines.

PubMed

Schmid-Burgk, Jonathan L; Schmidt, Tobias; Gaidt, Moritz M; Pelka, Karin; Latz, Eicke; Ebert, Thomas S; Hornung, Veit

2014-10-01

The application of designer nucleases allows the induction of DNA double-strand breaks (DSBs) at user-defined genomic loci. Due to imperfect DNA repair mechanisms, DSBs can lead to alterations in the genomic architecture, such as the disruption of the reading frame of a critical exon. This can be exploited to generate somatic knockout cell lines. While high genome editing activities can be achieved in various cellular systems, obtaining cell clones that contain all-allelic frameshift mutations at the target locus of interest remains a laborious task. To this end, we have developed an easy-to-follow deep sequencing workflow and the evaluation tool OutKnocker (www.OutKnocker.org), which allows convenient, reliable, and cost-effective identification of knockout cell lines. © 2014 Schmid-Burgk et al.; Published by Cold Spring Harbor Laboratory Press.

The U.S. Environmental Protection Industry: The Technical Document (1995)

EPA Pesticide Factsheets

This 1995 report provides a detailed explanation of the data sources and procedures used to generate in the estimates presented in The U.S. Environmental Protection Industry: A Proposed Framework for Assessment.

77 FR 59206 - U.S. Customs and Border Protection

Federal Register 2010, 2011, 2012, 2013, 2014

2012-09-26

... collection techniques or the use of other forms of information technology; and (e) the annual cost burden to... Schedule of the United States (HTSUS). This declaration includes information such as the quantity, value... DEPARTMENT OF HOMELAND SECURITY U.S. Customs and Border Protection Agency Information Collection...

S-protected thiolated chitosan: Synthesis and in vitro characterization

PubMed Central

Dünnhaupt, Sarah; Barthelmes, Jan; Thurner, Clemens C.; Waldner, Claudia; Sakloetsakun, Duangkamon; Bernkop-Schnürch, Andreas

2012-01-01

Purpose of the present study was the generation and evaluation of novel thiolated chitosans, so-named S-protected thiolated chitosans as mucosal drug delivery systems. Stability of all conjugates concerning swelling and disintegration behavior as well as drug release was examined. Mucoadhesive properties were evaluated in vitro on intestinal mucosa. Different thiolated chitosans were generated displaying increasing amounts of attached free thiol groups on the polymer, whereby more than 50% of these thiol groups were linked with 6-mercaptonicotinamide. Based on the implementation of this hydrophobic residue, the swelling behavior was 2-fold decreased, whereas stability was essentially improved. Their mucoadhesive properties were 2- and 14-fold increased compared to corresponding thiolated and unmodified chitosans, respectively. Release studies out of matrix tablets comprising the novel conjugates revealed a controlled release of a model peptide. Accordingly, S-protected thiomers represent a promising type of mucoadhesive polymers for the development of various mucosal drug delivery systems. PMID:22839999

S-protected thiolated chitosan: synthesis and in vitro characterization.

PubMed

Dünnhaupt, Sarah; Barthelmes, Jan; Thurner, Clemens C; Waldner, Claudia; Sakloetsakun, Duangkamon; Bernkop-Schnürch, Andreas

2012-10-01

Purpose of the present study was the generation and evaluation of novel thiolated chitosans, so-named S-protected thiolated chitosans as mucosal drug delivery systems. Stability of all conjugates concerning swelling and disintegration behavior as well as drug release was examined. Mucoadhesive properties were evaluated in vitro on intestinal mucosa. Different thiolated chitosans were generated displaying increasing amounts of attached free thiol groups on the polymer, whereby more than 50% of these thiol groups were linked with 6-mercaptonicotinamide. Based on the implementation of this hydrophobic residue, the swelling behavior was 2-fold decreased, whereas stability was essentially improved. Their mucoadhesive properties were 2- and 14-fold increased compared to corresponding thiolated and unmodified chitosans, respectively. Release studies out of matrix tablets comprising the novel conjugates revealed a controlled release of a model peptide. Accordingly, S-protected thiomers represent a promising type of mucoadhesive polymers for the development of various mucosal drug delivery systems. Copyright © 2012 Elsevier Ltd. All rights reserved.

Block Copolymer Micellization as a Protection Strategy for DNA Origami.

PubMed

Agarwal, Nayan P; Matthies, Michael; Gür, Fatih N; Osada, Kensuke; Schmidt, Thorsten L

2017-05-08

DNA nanotechnology enables the synthesis of nanometer-sized objects that can be site-specifically functionalized with a large variety of materials. For these reasons, DNA-based devices such as DNA origami are being considered for applications in molecular biology and nanomedicine. However, many DNA structures need a higher ionic strength than that of common cell culture buffers or bodily fluids to maintain their integrity and can be degraded quickly by nucleases. To overcome these deficiencies, we coated several different DNA origami structures with a cationic poly(ethylene glycol)-polylysine block copolymer, which electrostatically covered the DNA nanostructures to form DNA origami polyplex micelles (DOPMs). This straightforward, cost-effective, and robust route to protect DNA-based structures could therefore enable applications in biology and nanomedicine where unprotected DNA origami would be degraded. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease

PubMed Central

Sabzehei, Faezeh; Kouhpayeh, Shirin; Dastjerdeh, Mansoureh Shahbazi; Khanahmad, Hossein; Salehi, Rasoul; Naderi, Shamsi; Taghizadeh, Razieh; Rabiei, Parisa; Hejazi, Zahra; Shariati, Laleh

2017-01-01

Background: 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. Materials and 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. Results: 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). Conclusion: 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. PMID:29285485

In vivo topography of Rap1p-DNA complex at Saccharomyces cerevisiae TEF2 UAS(RPG) during transcriptional regulation.

PubMed

De Sanctis, Veronica; La Terra, Sabrina; Bianchi, Alessandro; Shore, David; Burderi, Luciano; Di Mauro, Ernesto; Negri, Rodolfo

2002-04-26

We have analyzed in detail the structure of RAP1-UAS(RPG) complexes in Saccharomyces cerevisiae cells using multi-hit KMnO(4), UV and micrococcal nuclease high-resolution footprinting. Three copies of the Rap1 protein are bound to the promoter simultaneously in exponentially growing cells, as shown by KMnO(4) multi-hit footprinting analysis, causing extended and diagnostic changes in the DNA structure of the region containing the UAS(RPG). Amino acid starvation does not cause loss of Rap1p from the complex; however, in vivo UV-footprinting reveals the occurrence of structural modifications of the complex. Moreover, low-resolution micrococcal nuclease digestion shows that the chromatin of the entire region is devoid of positioned nucleosomes but is susceptible to changes in accessibility to the nuclease upon amino acid starvation. The implications of these results for the mechanism of Rap1p action are discussed. (c) 2002 Elsevier Science Ltd.

Grumman S2F-1 Tracker at NACA Lewis

NASA Image and Video Library

1956-08-21

The NACA’s Lewis Flight Propulsion Laboratory acquired the Grumman S2F-1 Tracker from the Navy in 1955 to study icing instrumentation. Lewis’s icing research program was winding down at the time. The use of jet engines was increasing thus reducing the threat of ice accumulation. Nonetheless Lewis continued research on the instrumentation used to detect icing conditions. The S2F-1 Tracker was a carrier-based submarine hunter for the Navy. Grumman developed the Tracker as a successor to its Korean War-era Guardian patrol aircraft. Prototypes first flew in late 1952 and battle-ready versions entered Naval service in early 1954. The Navy utilized the Trackers to protect fleets from attack.

Silymarin preconditioning protected insulin resistant rats from liver ischemia-reperfusion injury: role of endogenous H2S.

PubMed

Younis, Nahla N; Shaheen, Mohamed A; Mahmoud, Mona F

2016-08-01

Hydrogen sulfide (H2S) can protect against hepatic ischemia-reperfusion injury (HIR). However, it is unknown whether it can protect against HIR in insulin resistance. This study investigated the protective effects of silymarin against HIR in a rat model of insulin resistance and the possible involvement of endogenous H2S. Insulin resistance was first established using 10% fructose in drinking water for 10 weeks. HIR was conducted in fructose-fed rats treated with saline or silymarin (100 mg/kg), 15 min before HIR (30 min ischemia, followed by 1 h reperfusion). Insulin resistance and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), total nitrites (NO2(-)), and H2S were measured. Hepatic malondialdehyde (MDA), reduced glutathione (GSH), hydroxyproline, H2S synthesizing activity, and mRNA expression of cystathionine β-synthase (CBS) or cystathionine γ-lyase (CSE) were determined. Additionally, histopathological examination involved H&E, Sirius red, and caspase-3 immunostaining. Fructose-induced insulin resistance increased serum ALT, TNF-α, H2S and H2S synthesizing activity, and hepatic MDA, hydroxyproline, and CSE mRNA and decreased NO2(-) and GSH. These changes exacerbated the HIR injury in which endogenous H2S production was auxiliary increased. Silymarin preconditioning decreased ALT, AST, MDA, NO2(-), TNF-α, and TNF-α/IL-10 ratio, increased GSH, IL-10, improved hepatic architecture, and lowered caspase-3 immunostaining. Serum H2S, its hepatic synthesizing activity, and CSE and CBS mRNA expressions were all suppressed by silymarin pretreatment. The increases in endogenous H2S exacerbate HIR injury, whereas silymarin preconditioning protected against HIR in insulin resistant rats via powerful antioxidant, anti-inflammatory, and antiapoptotic effects along with suppressing H2S production. Copyright © 2016 Elsevier Inc. All rights reserved.

Conservation plan for protected species on Naval Petroleum Reserve No. 1, Kern County, California

DOE Office of Scientific and Technical Information (OSTI.GOV)

Otten, M.R.M.; Cypher, B.L.

1997-07-01

Habitats in and around Naval Petroleum Reserve No. 1 (NPR-1) support populations of various vertebrates and plants, including a number of threatened and endangered species. Adequate conservation of habitats and species, particularly protected species, can be facilitated through development and implementation of management plans. This document provides a comprehensive plan for the conservation of protected species on NPR-1, through compliance with terms and conditions expressed in Biological Opinions rendered by the U.S. Fish and Wildlife Service for NPR-1 activities. Six conservation strategies by which threatened and endangered species have been, and will be, protected are described: population monitoring, mitigation strategies,more » special studies, operating guidelines and policies, information transfer and outreach, and the endangered species conservation area. Population monitoring programs are essential for determining population densities and for assessing the effects of oil field developments and environmental factors on protected species. Mitigation strategies (preactivity surveys and habitat reclamation) are employed to minimize the loss of important habitats components and to restore previously disturbed lands to conditions more suitable for species` use. A number of special studies were undertaken between 1985 and 1995 to investigate the effectiveness of a variety of population and habitat management techniques with the goal of increasing the density of protected species. Operating guidelines and policies governing routine oil field activities continue to be implemented to minimize the potential for the incidental take of protected species and minimize damage to wildlife habitats. Information transfer and outreach activities are important means by which technical and nontechnical information concerning protected species conservation on NPR-1 is shared with both the scientific and non-scientific public.« less

Should the U.S. Department of State Continue to Use Private Security Contractors to Protect U.S. Diplomats

DTIC Science & Technology

2008-06-13

USE PRIVATE SECURITY CONTRACTORS TO PROTECT U.S. DIPLOMATS 5c. PROGRAM ELEMENT NUMBER 5d. PROJECT NUMBER 5e. TASK NUMBER 6. AUTHOR( S ...Special Agent Derek Dela-Cruz 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME( S ) AND ADDRESS(ES) U.S. Army Command and General Staff College...

Involvement of miR-1 in the protective effect of hydrogen sulfide against cardiomyocyte apoptosis induced by ischemia/reperfusion.

PubMed

Kang, Bo; Hong, Jiang; Xiao, Jian; Zhu, Xiaoyan; Ni, Xin; Zhang, Yufeng; He, Bin; Wang, Zhinong

2014-10-01

The protective effect of hydrogen sulfide (H2S) against myocardial ischemia/reperfusion (IR) injury via anti-apoptotic signaling is well established, but the underlying mechanism remains unclear. Recently, miRNAs have been identified as important mediators of myocardial injury by regulating apoptosis-related genes. It was found in our previous preliminary study that microRNA-1 (miR-1) expression underwent a significant change in IR group compared to H2S preconditioned group, indicating that miR-1 possessed myocyte-specific properties. In the present study, we intended to see whether miR-1 participated in H2S protection of cardiomyocytes against IR-induced apoptosis by regulating apoptosis-related genes. Cardiomyocytes of neonatal rats were subjected to hypoxia/reoxygenation (HR) injury with or without H2S preconditioning, while the myocardium of adult SD rats was subjected to IR with or without H2S preconditioning. It was found that HR injury increased apoptosis of cardiac myocytes, up-regulated the expression of miR-1, and down-regulated the expression of Bcl-2. H2S preconditioning attenuated cardiomyocyte apoptosis and LDH release, as well as enhanced cell viability following HR injury. MiR-1 was up-regulated by HR and down-regulated by H2S preconditioning. In contrast, Bcl-2 was down-regulated by HR and up-regulated by H2S preconditioning. In addition, Bcl-2 protein was down-regulated by the miR-1 mimic in a dose-dependent manner. H2S also attenuated IR-induced cardiomyocyte apoptosis in vivo. MiR-1 regulated H2S protection of cardiomyocytes against IR-induced apoptosis by stimulating Bcl-2. These results implicate miR-1 as an important regulator of H2S on the IR myocardium.

Audit of the management and cost of the Department of Energy`s protective forces

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1994-07-01

The Department of Energy`s safeguards and security program is designed to provide appropriate, efficient, and effective protection of the Department`s nuclear weapons, nuclear materials, facilities, and classified information. These items must be protected against theft, sabotage, espionage, and terrorist activity, with continuing emphasis on protection against the insider threat. The purpose of the audit was to determine if protective forces were efficiently managed and appropriately sized in light of the changing missions and current budget constraints. The authors found that the cost of physical security at some sites had grown beyond those costs incurred when the site was in fullmore » production. This increase was due to a combination of factors, including concerns about the adequacy of physical security, reactions to the increase in terrorism in the early 1980s with the possibility of hostile attacks, and the selection of security system upgrades without adequate consideration of cost effectiveness. Ongoing projects to upgrade security systems were not promptly reassessed when missions changed and levels of protection were not determined in a way which considered the attractiveness of the material being protected. The authors also noted several opportunities for the Department to improve the operational efficiency of its protective force operations, including, eluminating overtime paid to officers prior to completion of the basic 40-hour workweek, paying hourly wages of unarmed guards which are commensurate with their duties, consolidating protective force units, transferring law enforcement duties to local law agencies, eliminating or reducing paid time to exercise, and standardizing supplies and equipment used by protective force members.« less

Genome editing strategies: potential tools for eradicating HIV-1/AIDS

PubMed Central

Khalili, Kamel; Gordon, Jennifer; Cosentino, Laura; Hu, Wenhui

2015-01-01

Current therapy for controlling HIV-1 infection and preventing AIDS progression has profoundly decreased viral replication in cells susceptible to HIV-1 infection, but it does not eliminate the low level of viral replication in latently infected cells which contain integrated copies of HIV-1 proviral DNA. There is an urgent need for the development of HIV-1 genome eradication strategies that will lead to a permanent or “sterile” cure of HIV-1/AIDS. In the past few years, novel nuclease-initiated genome editing tools have been developing rapidly, including ZFNs, TALENs, and the CRISPR/Cas9 system. These surgical knives, which can excise any genome, provide a great opportunity to eradicate the HIV-1 genome by targeting highly conserved regions of the HIV-1 long terminal repeats or essential viral genes. Given the time consuming and costly engineering of target-specific ZFNs and TALENs, the RNA-guided endonuclease Cas9 technology has emerged as a simpler and more versatile technology to allow permanent removal of integrated HIV-1 proviral DNA in eukaryotic cells, and hopefully animal models or human patients. The major unmet challenges of this approach at present include inefficient nuclease gene delivery, potential off-target cleavage, and cell-specific genome targeting. Nanoparticle or lentivirus-mediated delivery of next generation Cas9 technologies including nickase or RNA-guided FokI nuclease (RFN) will further improve the potential for genome editing to become a promising approach for curing HIV-1/AIDS. PMID:25716921

U.S. Environmental Protection Agency, Region 6 National Priorities List (NPL) Boundaries - 05/12/2014

EPA Pesticide Factsheets

Boundaries of sites in U.S. Environmental Protection Agency, Region 6 which are documented as being part of the National Priorities List as of May 12, 2014. The locations were determined by U.S. Environmental Protection Agency, Region 6 Superfund RPMs.

FAN1 acts with FANCI-FANCD2 to promote DNA interstrand cross-link repair.

PubMed

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.

Hanford Site Groundwater Protection Management Program: Revision 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

Groundwater protection is a national priority that is promulgated in a variety of environmental regulations at local, state, and federal levels. To effectively coordinate and ensure compliance with applicable regulations, the US Department of Energy has issued DOE Order 5400.1 (now under revision) that requires all US Department of Energy facilities to prepare separate groundwater protection program descriptions and plans. This document describes the Groundwater Protection Management Program for the Hanford Site located in the state of Washington. DOE Order 5400.1 specifies that the Groundwater Protection Management Program cover the following general topical areas: (1) documentation of the groundwater regime,more » (2) design and implementation of a groundwater monitoring program to support resource management and comply with applicable laws and regulations, (3) a management program for groundwater protection and remediation, (4) a summary and identification of areas that may be contaminated with hazardous waste, (5) strategies for controlling these sources, (6) a remedial action program, and (7) decontamination and decommissioning and related remedial action requirements. Many of the above elements are covered by existing programs at the Hanford Site; thus, one of the primary purposes of this document is to provide a framework for coordination of existing groundwater protection activities. Additionally, it describes how information needs are identified and can be incorporated into existing or proposed new programs. The Groundwater Protection Management Program provides the general scope, philosophy, and strategies for groundwater protection/management at the Hanford Site. Subtier documents provide the detailed plans for implementing groundwater-related activities and programs. Related schedule and budget information are provided in the 5-year plan for environmental restoration and waste management at the Hanford Site.« less

Protective effect of esculin against prooxidant aflatoxin B1-induced nephrotoxicity in mice.

PubMed

Naaz, Farah; Abdin, M Z; Javed, Saleem

2014-02-01

The study was designed to investigate the protective effect of esculin against pro-oxidant aflatoxin B1 (AFB1)-induced nephrotoxicity in mice. In this study toxicity was developed by oral administration of AFB1 at a dose of 66.60 μg/kg bw/day for 90 days in male Swiss albino mice. Esculin (150 mg/kg bw/0.2 ml/day) and standard compound ascorbic acid (300 mg/kg bw/0.2 ml/day) was given after 30 min of AFB1 administration for 90 days. Protective efficacy was assessed by measuring the levels of lipid peroxidation (LPO) and non-enzymatic antioxidants such as reduced glutathione (GSH) and also by measuring activities of enzymatic antioxidants such as glutathione peroxidase (GPX), glutathione-S-transferase (GST), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT) in kidney. Results were analysed at the 30(th), 60(th) and 90(th) day of the daily treatments, which showed a decrease in the level of LPO and an increase in the levels of enzymatic and non-enzymatic antioxidants. The protective effect of esculin was further proved by histopathological findings as it exhibited regenerative activities in mice renal tubules against AFB1-induced nephrotoxicity. The results obtained clearly demonstrate that the protective efficacy of esculin against pro-oxidant AFB1-induced nephrotoxicity in mice might be due to its antioxidants and free radical scavenging properties.

Protective glove use and hygiene habits modify the associations of specific pesticides with Parkinson’s disease

PubMed Central

Furlong, Melissa; Tanner, Caroline M; Goldman, Samuel M; Bhudhikanok, Grace S.; Blair, Aaron; Chade, Anabel; Comyns, Kathleen; Hoppin, Jane A.; Kasten, Meike; Korell, Monica; Langston, J William; Marras, Connie; Meng, Cheryl; Richards, Marie; Ross, G Webster; Umbach, David M; Sandler, Dale P; Kamel, Freya

2014-01-01

Pesticides have been associated with Parkinson’s disease (PD), and protective gloves and workplace hygiene can reduce pesticide exposure. We assessed whether use of gloves and workplace hygiene modified associations between pesticides and PD. The Farming and Movement Evaluation (FAME) Study is a nested case-control study within the Agricultural Health Study. Use of protective gloves, other PPE, and hygiene practices were determined by questionnaire (69 cases and 237 controls were included). We considered interactions of gloves and hygiene with ever-use of pesticides for all pesticides with ≥ 5 exposed and unexposed cases and controls in each glove-use stratum (paraquat, permethrin, rotenone, and trifluralin). 61% of respondents consistently used protective gloves and 87% consistently used ≥ 2 hygiene practices. Protective glove use modified the associations of paraquat and permethrin with PD: neither pesticide was associated with PD among protective glove users, while both pesticides were associated with PD among non-users (paraquat OR 3.9 [95% CI 1.3, 11.7], interaction p=0.15; permethrin OR 4.3 [95% CI 1.2, 15.6] interaction p=0.05). Rotenone was associated with PD regardless of glove use. Trifluralin was associated with PD among people who used <2 hygiene practices (OR 5.5 [95% CI 1.1, 27.1]) but was not associated with PD among people who used 2 or more practices (interaction p=0.02). Although sample size was limited in the FAME study, protective glove use and hygiene practices appeared to be important modifiers of the association between pesticides and PD and may reduce risk of PD associated with certain pesticides. PMID:25461423

Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells*♦

PubMed Central

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

Osteoblasts Protect AML Cells from SDF-1-Induced Apoptosis

PubMed Central

Kremer, Kimberly N.; Dudakovic, Amel; McGee-Lawrence, Meghan E.; Philips, Rachael L.; Hess, Allan D.; Smith, B. Douglas; van Wijnen, Andre J.; Karp, Judith E.; Kaufmann, Scott H.; Westendorf, Jennifer J.; Hedin, Karen E.

2014-01-01

The bone marrow provides a protective environment for acute myeloid leukemia (AML) cells that often allows leukemic stem cells to survive standard chemotherapeutic regimens. Targeting these leukemic stem cells within the bone marrow is critical for preventing relapse. We recently demonstrated that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis in AML cell lines and in patient samples expressing high levels of its receptor, CXCR4. Here we show that a subset of osteoblast lineage cells within the bone marrow can protect AML cells from undergoing apoptosis in response to the SDF-1 naturally present in that location. In co-culture systems, osteoblasts at various stages of differentiation protected AML cell lines and patient isolates from SDF-1-induced apoptosis. The differentiation of the osteoblast cell lines, MC3T3 and W-20-17, mediated this protection via a cell contact-independent mechanism. In contrast, bone marrow-derived mesenchymal cells, the precursors of osteoblasts, induced apoptosis in AML cells via a CXCR4-dependent mechanism and failed to protect AML cells from exogenously added SDF-1. These results indicate that osteoblasts in the process of differentiation potently inhibit the SDF-1-driven apoptotic pathway of CXCR4-expressing AML cells residing in the bone marrow. Drugs targeting this protective mechanism could potentially provide a new approach to treating AML by enhancing the SDF-1-induced apoptosis of AML cells residing within the bone marrow microenvironment. PMID:24851270

U.S. Environmental Protection Agency, Region 6 National Priorities List (NPL) Sites - 05/12/2014

EPA Pesticide Factsheets

Point locations for sites in U.S. Environmental Protection Agency, Region 6 which are documented as being part of the National Priorities List as of May 12, 2014. The locations were determined by U.S. Environmental Protection Agency, Region 6 Superfund RPMs.

Diabetes Mellitus Is Associated With Reduced High-Density Lipoprotein Sphingosine-1-Phosphate Content and Impaired High-Density Lipoprotein Cardiac Cell Protection.

PubMed

Brinck, Jonas W; Thomas, Aurélien; Lauer, Estelle; Jornayvaz, François R; Brulhart-Meynet, Marie-Claude; Prost, Jean-Christophe; Pataky, Zoltan; Löfgren, Patrik; Hoffstedt, Johan; Eriksson, Mats; Pramfalk, Camilla; Morel, Sandrine; Kwak, Brenda R; van Eck, Miranda; James, Richard W; Frias, Miguel A

2016-05-01

The dyslipidemia of type 2 diabetes mellitus has multiple etiologies and impairs lipoprotein functionality, thereby increasing risk for cardiovascular disease. High-density lipoproteins (HDLs) have several beneficial effects, notably protecting the heart from myocardial ischemia. We hypothesized that glycation of HDL could compromise this cardioprotective effect. We used in vitro (cardiomyocytes) and ex vivo (whole heart) models subjected to oxidative stress together with HDL isolated from diabetic patients and nondiabetic HDL glycated in vitro (methylglyoxal). Diabetic and in vitro glycated HDL were less effective (P<0.05) than control HDL in protecting from oxidative stress. Protection was significantly, inversely correlated with the degree of in vitro glycation (P<0.001) and the levels of hemoglobin A1c in diabetic patients (P<0.007). The ability to activate protective, intracellular survival pathways involving Akt, Stat3, and Erk1/2 was significantly reduced (P<0.05) using glycated HDL. Glycation reduced the sphingosine-1-phosphate (S1P) content of HDL, whereas the S1P concentrations of diabetic HDL were inversely correlated with hemoglobin A1c (P<0.005). The S1P contents of in vitro glycated and diabetic HDL were significantly, positively correlated (both <0.01) with cardiomyocyte survival during oxidative stress. Adding S1P to diabetic HDL increased its S1P content and restored its cardioprotective function. Our data demonstrate that glycation can reduce the S1P content of HDL, leading to increased cardiomyocyte cell death because of less effective activation of intracellular survival pathways. It has important implications for the functionality of HDL in diabetes mellitus because HDL-S1P has several beneficial effects on the vasculature. © 2016 American Heart Association, Inc.

THE U.S. ENVIRONMENTAL PROTECTION AGENCY VISUAL PLUMES MODELING SOFTWARE

EPA Science Inventory

The U.S. Environmental Protection Agency's Center for Exposure Assessment Modeling (CEAM) at the Ecosystems Research Division in Athens, Georgia develops environmental exposure models, including plume models, and provides technical assistance to model users. The mixing zone and f...

Vaccination with Leishmania histone H1-pulsed dendritic cells confers protection in murine visceral leishmaniasis.

PubMed

Agallou, Maria; Smirlis, Despina; Soteriadou, Ketty P; Karagouni, Evdokia

2012-07-20

Visceral leishmaniasis is the most severe form of leishmaniases affecting millions of people worldwide often resulting in death despite optimal therapy. Thus, there is an urgent need for the development of effective anti-infective vaccine(s). In the present study, we evaluated the prophylactic value of bone marrow-derived dendritic cells (BM-DCs) pulsed with the Leishmania (L.) infantum histone H1. We developed fully mature BM-DCs characterized by enhanced capacity of IL-12 production after ex vivo pulsing with GST-LeishH1. Intravenous administration of these BM-DCs in naive BALB/c mice resulted in antigen-specific spleenocyte proliferation and IgG1 isotype antibody production and conferred protection against experimental challenge with L. infantum independently of CpG oligonucleotides (ODNs) co-administration. Protection was associated with a pronounced enhancement of parasite-specific IFNγ-producing cells and reduction of cells producing IL-10, whereas IL-4 production was comparable in protected and non-protected mice. The polarization of immune responses to Th1 type was further confirmed by the elevation of parasite-specific IgG2a/IgG1 ratio in protected mice. The above data indicate the immunostimulatory capacity of Leishmania histone H1 and further support its exploitation as a candidate protein for vaccine development against leishmaniasis. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

PubMed

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.

A Single Immunization with Soluble Recombinant Trimeric Hemagglutinin Protects Chickens against Highly Pathogenic Avian Influenza Virus H5N1

PubMed Central

Cornelissen, Lisette A. H. M.; de Vries, Robert P.; de Boer-Luijtze, Els A.; Rigter, Alan; Rottier, Peter J. M.; de Haan, Cornelis A. M.

2010-01-01

Background The highly pathogenic avian influenza (HPAI) virus H5N1 causes multi-organ disease and death in poultry, resulting in significant economic losses in the poultry industry. In addition, it poses a major public health threat as it can be transmitted directly from infected poultry to humans with very high (60%) mortality rate. Effective vaccination against HPAI H5N1 would protect commercial poultry and would thus provide an important control measure by reducing the likelihood of bird-to-bird and bird-to-human transmission. Methodology/Principal Findings In the present study we evaluated the vaccine potential of recombinant soluble trimeric subtype 5 hemagglutinin (sH53) produced in mammalian cells. The secreted, purified sH53 was biologically active as demonstrated by its binding to ligands in a sialic acid-dependent manner. It was shown to protect chickens, in a dose-dependent manner, against a lethal challenge with H5N1 after a single vaccination. Protected animals did not shed challenge virus as determined by a quantitative RT-PCR on RNA isolated from trachea and cloaca swabs. Also in mice, vaccination with sH53 provided complete protection against challenge with HPAI H5N1. Conclusions/Significance Our results demonstrate that sH53 constitutes an attractive vaccine antigen for protection of chickens and mammals against HPAI H5N1. As these recombinant soluble hemagglutinin preparations can be produced with high yields and with relatively short lead time, they enable a rapid response to circulating and potentially pandemic influenza viruses. PMID:20498717

VEZF1 Elements Mediate Protection from DNA Methylation

PubMed Central

Strogantsev, Ruslan; Gaszner, Miklos; Hair, Alan; Felsenfeld, Gary; West, Adam G.

2010-01-01

There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm β-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state. PMID:20062523

Development of a 5'-nuclease real-time PCR assay targeting fliP for the rapid identification of Burkholderia mallei in clinical samples.

PubMed

Tomaso, Herbert; Scholz, Holger C; Al Dahouk, Sascha; Eickhoff, Meike; Treu, Thomas M; Wernery, Renate; Wernery, Ulrich; Neubauer, Heinrich

2006-02-01

Burkholderia mallei is a potential biological agent that causes glanders or farcy in solipeds, a disease notifiable to the Office International des Epizooties (OIE). The number of reported outbreaks has increased steadily during the last decade, but diagnosis is hampered by the low bacterial load in infected tissues and excretions. We developed a B. mallei-specific 5'-nuclease real-time PCR assay that targets the fliP gene of B. mallei and includes an internal amplification control. Specificity was assessed with 19 B. mallei strains, 27 Burkholderia pseudomallei strains, other Burkholderia strains of 29 species, and clinically relevant non-Burkholderia organisms. Amplification products were observed in all B. mallei strains but in no other bacteria. The linear range of the B. mallei real-time PCR covered concentrations from 240 pg to 70 fg of bacterial DNA/reaction. The detection limit was 60 fg of B. mallei DNA. The clinical applicability of the assay was demonstrated by use of organ samples from diseased horses of a recent outbreak that was reported to the OIE by the United Arab Emirates in 2004. Compared with conventional PCR, our rapid 5'-nuclease real-time PCR assay for the specific identification of B. mallei has a lower risk of carryover contamination and eliminates the need for post-PCR manipulations. This real-time PCR assay also shortens the turnaround time for results and has the potential for automation.

Hypothalamic S1P/S1PR1 axis controls energy homeostasis.

PubMed

Silva, Vagner R R; Micheletti, Thayana O; Pimentel, Gustavo D; Katashima, Carlos K; Lenhare, Luciene; Morari, Joseane; Mendes, Maria Carolina S; Razolli, Daniela S; Rocha, Guilherme Z; de Souza, Claudio T; Ryu, Dongryeol; Prada, Patrícia O; Velloso, Lício A; Carvalheira, José B C; Pauli, José Rodrigo; Cintra, Dennys E; Ropelle, Eduardo R

2014-09-25

Sphingosine 1-phosphate receptor 1 (S1PR1) is a G-protein-coupled receptor for sphingosine-1-phosphate (S1P) that has a role in many physiological and pathophysiological processes. Here we show that the S1P/S1PR1 signalling pathway in hypothalamic neurons regulates energy homeostasis in rodents. We demonstrate that S1PR1 protein is highly enriched in hypothalamic POMC neurons of rats. Intracerebroventricular injections of the bioactive lipid, S1P, reduce food consumption and increase rat energy expenditure through persistent activation of STAT3 and the melanocortin system. Similarly, the selective disruption of hypothalamic S1PR1 increases food intake and reduces the respiratory exchange ratio. We further show that STAT3 controls S1PR1 expression in neurons via a positive feedback mechanism. Interestingly, several models of obesity and cancer anorexia display an imbalance of hypothalamic S1P/S1PR1/STAT3 axis, whereas pharmacological intervention ameliorates these phenotypes. Taken together, our data demonstrate that the neuronal S1P/S1PR1/STAT3 signalling axis plays a critical role in the control of energy homeostasis in rats.

STATUS OF THE U.S. ENVIRONMENTAL PROTECTION AGENCY'S ENCODRINE DISRUPTOR SCREENING PROGRAM

EPA Science Inventory

Status of the U.S. Environmental Protection Agency's Endocrine Disruptor Screening Program. Susan Laws. Endocrinology Branch, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, ORD, U.S. EPA, RTP, NC.

In response to emergi...

CD44S-hyaluronan interactions protect cells resulting from EMT against anoikis

PubMed Central

Cieply, Benjamin; Koontz, Colton; Frisch, Steven M.

2016-01-01

The detachment of normal epithelial cells from matrix triggers an apoptotic response known as anoikis, during homeostatic turnover. Metastatic tumor cells evade anoikis, by mechanisms that are only partly characterized. In particular, the epithelial–mesenchymal transition (EMT) in a subset of invasive tumor cells confers anoikis-resistance. In some cases, EMT up-regulates the cancer stem cell marker CD44S and the enzyme hyaluronic acid synthase-2 (HAS2). CD44S is the major receptor for hyaluronan in the extracellular matrix. Herein, we demonstrate that CD44S, unlike the CD44E isoform expressed in normal epithelial cells, contributes to the protection against anoikis. This protection requires the interaction of CD44S with hyaluronan (HA). CD44S–HA interaction is proposed to play an important role in tumor metastasis through enhanced cell survival under detached conditions. PMID:25937513

Direct stacking of sequence-specific nuclease-induced mutations to produce high oleic and low linolenic soybean oil.

PubMed

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 %.

Protecting unauthorized immigrant mothers improves their children’s mental health

PubMed Central

Hainmueller, Jens; Lawrence, Duncan; Martén, Linna; Black, Bernard; Figueroa, Lucila; Hotard, Michael; Jiménez, Tomás R.; Mendoza, Fernando; Rodriguez, Maria I.; Swartz, Jonas J.; Laitin, David D.

2018-01-01

The United States is embroiled in a debate about whether to protect or deport its estimated 11 million unauthorized immigrants, but the fact that these immigrants are also parents to more than 4 million U.S.-born children is often overlooked. We provide causal evidence of the impact of parents’ unauthorized immigration status on the health of their U.S. citizen children. The Deferred Action for Childhood Arrivals (DACA) program granted temporary protection from deportation to more than 780,000 unauthorized immigrants. We used Medicaid claims data from Oregon and exploited the quasi-random assignment of DACA eligibility among mothers with birthdates close to the DACA age qualification cutoff. Mothers’ DACA eligibility significantly decreased adjustment and anxiety disorder diagnoses among their children. Parents’ unauthorized status is thus a substantial barrier to normal child development and perpetuates health inequalities through the intergenerational transmission of disadvantage. PMID:28860206

Site-specific cleavage of the transactivation response site of human immunodeficiency virus RNA with a tat-based chemical nuclease.

PubMed Central

Jayasena, S D; Johnston, B H

1992-01-01

tat, an essential transactivator of gene transcription in the human immunodeficiency virus (HIV), is believed to activate viral gene expression by binding to the transactivation response (TAR) site located at the 5' end of all viral mRNAs. The TAR element forms a stem-loop structure containing a 3-nucleotide bulge that is the site for tat binding and is required for transactivation. Here we report the synthesis of a site-specific chemical ribonuclease based on the TAR binding domain of the HIV type 1 (HIV-1) tat. A peptide consisting of this 24-amino acid domain plus an additional C-terminal cysteine residue was chemically synthesized and covalently linked to 1,10-phenanthroline at the cysteine residue. The modified peptide binds to TAR sequences of both HIV-1 and HIV-2 and, in the presence of cupric ions and a reducing agent, cleaves these RNAs at specific sites. Cleavage sites on TAR sequences are consistent with peptide binding to the 3-nucleotide bulge, and the relative displacement of cleavage sites on the two strands suggests peptide binding to the major groove of the RNA. These results and existing evidence of the rapid cellular uptake of tat-derived peptides suggest that chemical nucleases based on tat may be useful for inactivating HIV mRNA in vivo. Images PMID:1565648

Climate protection in Germany`s bilateral development co-operation with the People`s Republic of China

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schneider, A.

1996-12-31

For globally sustainable development to be achieved, three concerns are central: productive economic growth, social justice and ecological sustainability. Development co-operation supports the realisation of these three goals in partner countries by helping to alleviate poverty, promote economic growth through private-sector development and protect vital natural resources. The aim of globally sustainable development can only be achieved if industrial countries too implement necessary reforms and structural adjustments at every level. Co-operation efforts with partners must therefore be complemented by coherent policies at home. This is a matter of credibility, but also of developmental far-sightedness. Internal reforms in the industrial countriesmore » secure financial leeway for their providing foreign assistance in the longer term. Environmental and resource protection as a focal point of Germany`s development co-operation with the PRC aims to preserve vital natural resources, shape economic development in their partner countries in an ecologically sound manner and put China in a position to participate in global endeavours to protect the environment. Climate protection measures figure prominently in this area. This is justified given China`s share of global CO{sub 2} emissions and the potential for energy-saving measures and measures to increase power intensity. This potential is derived primarily from the possibility of using energy-efficient technologies, increasing the relatively low energy prices and making use of renewable sources of energy.« less

Strikingly higher interleukin (IL)-1alpha, IL-1beta and soluble interleukin-1 receptor antagonist (sIL-1RA) but similar IL-2, sIL-2R, IL-3, IL-4, IL-6, sIL-6R, IL-10, tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta and interferon IFN-gamma urine levels in healthy females compared to healthy males: protection against urinary tract injury?

PubMed

Sadeghi, M; Daniel, V; Naujokat, C; Weimer, R; Opelz, G

2005-11-01

The aim of this prospective study was to examine gender-related differences of cytokines in the plasma and urine of healthy individuals that might provide a clue concerning the lower rate of chronic renal diseases in females. Soluble interleukin-1 receptor antagonist (sIL-1RA), interleukin (IL)-1alpha, IL-1beta, IL-2, sIL-2R, IL-3, IL-4, IL-6, sIL-6R, IL-10, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta(2) and interferon (IFN)-gamma were determined using standard enzyme-linked immunosorbent assay (ELISA). Cytokine levels were determined in simultaneously obtained plasma and urine samples of 18 male and 28 female healthy members of our laboratory staff. Urine cytokine levels were studied three times at 1-month intervals. All individuals had a negative urine nitrite test and showed no symptoms of urinary tract infection (UTI). Plasma levels of all studied cytokines were similar in males and females (P = n.s.). However, females had significantly higher urine IL-1alpha (P < 0.0001; P < 0.0001; P < 0.0001) and sIL-1RA (P = 0.0001; P = 0.0003; P = 0.0002) than males at three and higher IL-1beta at one of the three investigations (P = 0.098; P = 0.003; P = 0.073). Urine levels of the other cytokines were similar in males and females. Higher urine levels of IL-1alpha, IL-1beta and sIL-1RA in females may result from stimulation of cells in the urinary tract. Increased sIL-1RA might block T lymphocyte activation. The elevated cytokines may play a role in the protection of the female urinary tract from certain renal diseases, such as pyelonephritis and other inflammatory and sclerotic kidney diseases.

Adenosine A1 receptor activation increases myocardial protein S-nitrosothiols and elicits protection from ischemia-reperfusion injury in male and female hearts

PubMed Central

Shao, Qin; Casin, Kevin M.; Mackowski, Nathan; Murphy, Elizabeth; Steenbergen, Charles

2017-01-01

Nitric oxide (NO) plays an important role in cardioprotection, and recent work from our group and others has implicated protein S-nitrosylation (SNO) as a critical component of NO-mediated protection in different models, including ischemic pre- and post-conditioning and sex-dependent cardioprotection. However, studies have yet to examine whether protein SNO levels are similarly increased with pharmacologic preconditioning in male and female hearts, and whether an increase in protein SNO levels, which is protective in male hearts, is sufficient to increase baseline protection in female hearts. Therefore, we pharmacologically preconditioned male and female hearts with the adenosine A1 receptor agonist N6-cyclohexyl adenosine (CHA). CHA administration prior to ischemia significantly improved functional recovery in both male and female hearts compared to baseline in a Langendorff-perfused heart model of ischemia-reperfusion injury (% of preischemic function ± SE: male baseline: 37.5±3.4% vs. male CHA: 55.3±3.2%; female baseline: 61.4±5.7% vs. female CHA: 76.0±6.2%). In a separate set of hearts, we found that CHA increased p-Akt and p-eNOS levels. We also used SNO-resin-assisted capture with LC-MS/MS to identify SNO proteins in male and female hearts, and determined that CHA perfusion induced a modest increase in protein SNO levels in both male (11.4%) and female (12.3%) hearts compared to baseline. These findings support a potential role for protein SNO in a model of pharmacologic preconditioning, and provide evidence to suggest that a modest increase in protein SNO levels is sufficient to protect both male and female hearts from ischemic injury. In addition, a number of the SNO proteins identified with CHA treatment were also observed with other forms of cardioprotective stimuli in prior studies, further supporting a role for protein SNO in cardioprotection. PMID:28493997

THE U.S. ENVIRONMENTAL PROTECTION AGENCY'S SITE EMERGING TECHNOLOGY PROGRAM

EPA Science Inventory

Under the SITE Emerging Technology Program, the U.S. Environmental Protection Agency is seeking to foster the further development of technol- ogies that have been successfully tested at bench-scale and are now ready for pilot-scale testing, prior to field- or full-scale demonstra...

26 CFR 1.1286-2 - Stripped inflation-protected debt instruments.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 26 Internal Revenue 11 2013-04-01 2013-04-01 false Stripped inflation-protected debt instruments... Losses § 1.1286-2 Stripped inflation-protected debt instruments. Stripped inflation-protected debt instruments. If a Treasury Inflation-Protected Security is stripped under the Department of the Treasury's...

26 CFR 1.1286-2 - Stripped inflation-protected debt instruments.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 11 2014-04-01 2014-04-01 false Stripped inflation-protected debt instruments... Losses § 1.1286-2 Stripped inflation-protected debt instruments. Stripped inflation-protected debt instruments. If a Treasury Inflation-Protected Security is stripped under the Department of the Treasury's...

TD-LTE Wireless Private Network QoS Transmission Protection

NASA Astrophysics Data System (ADS)

Zhang, Jianming; Cheng, Chao; Wu, Zanhong

With the commencement of construction of the smart grid, the demand power business for reliability and security continues to improve, the reliability transmission of power TD-LTE Wireless Private Network are more and more attention. For TD-LTE power private network, it can provide different QoS services according to the user's business type, to protect the reliable transmission of business. This article describes in detail the AF module of PCC in the EPC network, specifically introduces set up AF module station and QoS mechanisms in the EPS load, fully considers the business characteristics of the special power network, establishing a suitable architecture for mapping QoS parameters, ensuring the implementation of each QoS business. Through using radio bearer management, we can achieve the reliable transmission of each business on physical channel.

Limited Human Factors Assessment of the QuadGard Limb Protection System: U.S. Marine Corps Systems Command Limb Protection Program Overview (QuadGard Phases 4 and 5 Production Designs)

DTIC Science & Technology

2011-09-01

in calculating the ergonomics associated with ballistic protection. MARCORSYSCOM established three design requirements: (1) system compatibility...knob. The Velcro disengaged, as designed , to allow the wearer unimpeded leg movement. The control knob is used to adjust the driver’s seat height...QuadGard Phases IV and V Production Designs ) by Richard S. Bruno ARL-TR-5656 September 2011

Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice

PubMed Central

Tomimatsu, Nozomi; Mukherjee, Bipasha; Hardebeck, Molly Catherine; Ilcheva, Mariya; Camacho, Cristel Vanessa; Harris, Janelle Louise; Porteus, Matthew; Llorente, Bertrand; Khanna, Kum Kum; Burma, Sandeep

2014-01-01

Resection of DNA double-strand breaks (DSBs) is a pivotal step during which the choice between NHEJ and HR DNA repair pathways is made. While CDKs are known to control initiation of resection, their role in regulating long-range resection remains elusive. Here we show that CDKs 1/2 phosphorylate the long-range resection nuclease EXO1 at four C-terminal S/TP sites during S/G2 phases of the cell cycle. Impairment of EXO1 phosphorylation attenuates resection, chromosomal integrity, cell survival, and HR, but augments NHEJ upon DNA damage. In contrast, cells expressing phospho-mimic EXO1 are proficient in resection even after CDK inhibition and favor HR over NHEJ. Mutation of cyclin-binding sites on EXO1 attenuates CDK binding and EXO1 phosphorylation, causing a resection defect that can be rescued by phospho-mimic mutations. Mechanistically, phosphorylation of EXO1 augments its recruitment to DNA breaks possibly via interactions with BRCA1. In sum, phosphorylation of EXO1 by CDKs is a novel mechanism regulating repair pathway choice. PMID:24705021

Antarctica’s Protected Areas Are Inadequate, Unrepresentative, and at Risk

PubMed Central

Shaw, Justine D.; Terauds, Aleks; Riddle, Martin J.; Possingham, Hugh P.; Chown, Steven L.

2014-01-01

Antarctica is widely regarded as one of the planet's last true wildernesses, insulated from threat by its remoteness and declaration as a natural reserve dedicated to peace and science. However, rapidly growing human activity is accelerating threats to biodiversity. We determined how well the existing protected-area system represents terrestrial biodiversity and assessed the risk to protected areas from biological invasions, the region's most significant conservation threat. We found that Antarctica is one of the planet's least protected regions, with only 1.5% of its ice-free area formally designated as specially protected areas. Five of the distinct ice-free ecoregions have no specially designated areas for the protection of biodiversity. Every one of the 55 designated areas that protect Antarctica's biodiversity lies closer to sites of high human activity than expected by chance, and seven lie in high-risk areas for biological invasions. By any measure, including Aichi Target 11 under the Convention on Biological Diversity, Antarctic biodiversity is poorly protected by reserves, and those reserves are threatened. PMID:24936869

Mitochondria selective S-nitrosation by mitochondria-targeted S-nitrosothiol protects against post-infarct heart failure in mouse hearts.

PubMed

Methner, Carmen; Chouchani, Edward T; Buonincontri, Guido; Pell, Victoria R; Sawiak, Stephen J; Murphy, Michael P; Krieg, Thomas

2014-07-01

Recently it has been shown that the mitochondria-targeted S-nitrosothiol MitoSNO protects against acute ischaemia/reperfusion (IR) injury by inhibiting the reactivation of mitochondrial complex I in the first minutes of reperfusion of ischaemic tissue, thereby preventing free radical formation that underlies IR injury. However, it remains unclear how this transient inhibition of mitochondrial complex I-mediated free radicals at reperfusion affects the long-term recovery of the heart following IR injury. Here we determined whether the acute protection by MitoSNO at reperfusion prevented the subsequent development of post-myocardial infarction heart failure. Mice were subjected to 30 min left coronary artery occlusion followed by reperfusion and recovery over 28 days. MitoSNO (100 ng/kg) was applied 5 min before the onset of reperfusion followed by 20 min infusion (1 ng/kg/min). Infarct size and cardiac function were measured by magnetic resonance imaging (MRI) 24 h after infarction. MitoSNO-treated mice exhibited reduced infarct size and preserved function. In addition, MitoSNO at reperfusion improved outcome measures 28 days post-IR, including preserved systolic function (63.7 ±1.8% LVEF vs. 53.7 ± 2.1% in controls, P = 0.01) and tissue fibrosis. MitoSNO action acutely at reperfusion reduces infarct size and protects from post-myocardial infarction heart failure. Therefore, targeted inhibition of mitochondrial complex I in the first minutes of reperfusion by MitoSNO is a rational therapeutic strategy for preventing subsequent heart failure in patients undergoing IR injury. © 2014 The Authors. European Journal of Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.

Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction.

PubMed

Ying, Na; Ju, Chuanjing; Sun, Xiuwei; Li, Letian; Chang, Hongbiao; Song, Guangping; Li, Zhongyi; Wan, Jiayu; Dai, Enyong

2017-01-01

MicroRNAs (miRNAs) constitute novel biomarkers for various diseases. Accurate and quantitative analysis of miRNA expression is critical for biomedical research and clinical theranostics. In this study, a method was developed for sensitive and specific detection of miRNAs via dual signal amplification based on duplex specific nuclease (DSN) and hybridization chain reaction (HCR). A reporter probe (RP), comprising recognition sequence (3' end modified with biotin) for a target miRNA of miR-21 and capture sequence (5' end modified with Fam) for HCR product, was designed and synthesized. HCR was initiated by partial sequence of initiator probe (IP), the other part of which can hybridize with capture sequence of RP, and was assembled by hairpin probes modified with biotin (H1-bio and H2-bio). A miR-21 triggered cyclical DSN cleavage of RP, which was immobilized to a streptavidin (SA) coated magnetic bead (MB). The released Fam labeled capture sequence then hybridized with the HCR product to generate a detectable dsDNA. This polymer was then dropped on lateral flow strip and positive result was observed. The proposed method allowed quantitative sequence-specific detection of miR-21 (with a detection limit of 2.1 fM, S/N = 3) in a dynamic range from 100 fM to 100 pM, with an excellent ability to discriminate differences in miRNAs. The method showed acceptable testing recoveries for the determination of miRNAs in serum.

41 CFR 109-1.5107 - Physical protection of personal property.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 41 Public Contracts and Property Management 3 2012-01-01 2012-01-01 false Physical protection of personal property. 109-1.5107 Section 109-1.5107 Public Contracts and Property Management Federal Property...-INTRODUCTION 1.51-Personal Property Management Standards and Practices § 109-1.5107 Physical protection of...

41 CFR 109-1.5107 - Physical protection of personal property.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 41 Public Contracts and Property Management 3 2014-01-01 2014-01-01 false Physical protection of personal property. 109-1.5107 Section 109-1.5107 Public Contracts and Property Management Federal Property...-INTRODUCTION 1.51-Personal Property Management Standards and Practices § 109-1.5107 Physical protection of...

41 CFR 109-1.5107 - Physical protection of personal property.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 41 Public Contracts and Property Management 3 2010-07-01 2010-07-01 false Physical protection of personal property. 109-1.5107 Section 109-1.5107 Public Contracts and Property Management Federal Property...-INTRODUCTION 1.51-Personal Property Management Standards and Practices § 109-1.5107 Physical protection of...

41 CFR 109-1.5107 - Physical protection of personal property.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 41 Public Contracts and Property Management 3 2013-07-01 2013-07-01 false Physical protection of personal property. 109-1.5107 Section 109-1.5107 Public Contracts and Property Management Federal Property...-INTRODUCTION 1.51-Personal Property Management Standards and Practices § 109-1.5107 Physical protection of...

41 CFR 109-1.5107 - Physical protection of personal property.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 41 Public Contracts and Property Management 3 2011-01-01 2011-01-01 false Physical protection of personal property. 109-1.5107 Section 109-1.5107 Public Contracts and Property Management Federal Property...-INTRODUCTION 1.51-Personal Property Management Standards and Practices § 109-1.5107 Physical protection of...

Immune Protection against Lethal Fungal-Bacterial Intra-Abdominal Infections

PubMed Central

Lilly, Elizabeth A.; Ikeh, Melanie; Nash, Evelyn E.; Fidel, Paul L.

2018-01-01

ABSTRACT Polymicrobial intra-abdominal infections (IAIs) are clinically prevalent and cause significant morbidity and mortality, especially those involving fungi. Our laboratory developed a mouse model of IAI and demonstrated that intraperitoneal inoculation with Candida albicans or other virulent non-albicans Candida (NAC) species plus Staphylococcus aureus resulted in 70 to 80% mortality in 48 to 72 h due to robust local and systemic inflammation (sepsis). Surprisingly, inoculation with Candida dubliniensis or Candida glabrata with S. aureus resulted in minimal mortality, and rechallenge of these mice with lethal C. albicans/S. aureus (i.e., coninfection) resulted in >90% protection. The purpose of this study was to define requirements for C. dubliniensis/S. aureus-mediated protection and interrogate the mechanism of the protective response. Protection was conferred by C. dubliniensis alone or by killed C. dubliniensis plus live S. aureus. S. aureus alone was not protective, and killed S. aureus compromised C. dubliniensis-induced protection. C. dubliniensis/S. aureus also protected against lethal challenge by NAC plus S. aureus and could protect for a long-term duration (60 days between primary challenge and C. albicans/S. aureus rechallenge). Unexpectedly, mice deficient in T and B cells (Rag-1 knockouts [KO]) survived both the initial C. dubliniensis/S. aureus challenge and the C. albicans/S. aureus rechallenge, indicating that adaptive immunity did not play a role. Similarly, mice depleted of macrophages prior to rechallenge were also protected. In contrast, protection was associated with high numbers of Gr-1hi polymorphonuclear leukocytes (PMNLs) in peritoneal lavage fluid within 4 h of rechallenge, and in vivo depletion of Gr-1+ cells prior to rechallenge abrogated protection. These results suggest that Candida species can induce protection against a lethal C. albicans/S. aureus IAI that is mediated by PMNLs and postulated to be a unique form of

The Aurora kinase Ipl1 maintains the centromeric localization of PP2A to protect cohesin during meiosis.

PubMed

Yu, Hong-Guo; Koshland, Douglas

2007-03-26

Homologue segregation during the first meiotic division requires the proper spatial regulation of sister chromatid cohesion and its dissolution along chromosome arms, but its protection at centromeric regions. This protection requires the conserved MEI-S332/Sgo1 proteins that localize to centromeric regions and also recruit the PP2A phosphatase by binding its regulatory subunit, Rts1. Centromeric Rts1/PP2A then locally prevents cohesion dissolution possibly by dephosphorylating the protein complex cohesin. We show that Aurora B kinase in Saccharomyces cerevisiae (Ipl1) is also essential for the protection of meiotic centromeric cohesion. Coupled with a previous study in Drosophila melanogaster, this meiotic function of Aurora B kinase appears to be conserved among eukaryotes. Furthermore, we show that Sgo1 recruits Ipl1 to centromeric regions. In the absence of Ipl1, Rts1 can initially bind to centromeric regions but disappears from these regions after anaphase I onset. We suggest that centromeric Ipl1 ensures the continued centromeric presence of active Rts1/PP2A, which in turn locally protects cohesin and cohesion.

Protection conferred by a recombinant Marek’s disease virus that expresses the spike protein from infectious bronchitis virus in specific pathogen-free chicken

PubMed Central

2012-01-01

Background In many countries, the predominant field isolates of infectious bronchitis virus (IBV) have been classified as QX-like strains since 1996. However, no commercial vaccines that are specific for this type of IBV are currently available. Therefore, there is an urgent need to develop novel vaccines that prevent QX-like IBV infection. Results A recombinant Marek’s disease virus (MDV), rMDV-S1, that expresses the S1 subunit of the spike (S) protein from the QX-like infectious bronchitis virus (IBV) was constructed by inserting the IBV S1 gene into the genome of the CVI988/Rispens strain of MDV. Specific pathogen-free (SPF) chickens that were vaccinated with rMDV-S1 were protected when challenged with the QX-like IBV. They were observed to have mild clinical signs of disease, a short virus-shedding period and low mortality. Additionally, the rMDV-S1 conferred full protection to chickens against virulent MDV, as did the CVI988/Rispens strain. Conclusions Our results demonstrate that rMDV-S1 is an effective and promising recombinant vaccine for the prevention of QX-like IBV infection. PMID:22559869

2D MoS2 as an efficient protective layer for lithium metal anodes in high-performance Li-S batteries

NASA Astrophysics Data System (ADS)

Cha, Eunho; Patel, Mumukshu D.; Park, Juhong; Hwang, Jeongwoon; Prasad, Vish; Cho, Kyeongjae; Choi, Wonbong

2018-04-01

Among the candidates to replace Li-ion batteries, Li-S cells are an attractive option as their energy density is about five times higher ( 2,600 Wh kg-1). The success of Li-S cells depends in large part on the utilization of metallic Li as anode material. Metallic lithium, however, is prone to grow parasitic dendrites and is highly reactive to several electrolytes; moreover, Li-S cells with metallic Li are also susceptible to polysulfides dissolution. Here, we show that 10-nm-thick two-dimensional (2D) MoS2 can act as a protective layer for Li-metal anodes, greatly improving the performances of Li-S batteries. In particular, we observe stable Li electrodeposition and the suppression of dendrite nucleation sites. The deposition and dissolution process of a symmetric MoS2-coated Li-metal cell operates at a current density of 10 mA cm-2 with low voltage hysteresis and a threefold improvement in cycle life compared with using bare Li-metal. In a Li-S full-cell configuration, using the MoS2-coated Li as anode and a 3D carbon nanotube-sulfur cathode, we obtain a specific energy density of 589 Wh kg-1 and a Coulombic efficiency of 98% for over 1,200 cycles at 0.5 C. Our approach could lead to the realization of high energy density and safe Li-metal-based batteries.

2D MoS2 as an efficient protective layer for lithium metal anodes in high-performance Li-S batteries.

PubMed

Cha, Eunho; Patel, Mumukshu D; Park, Juhong; Hwang, Jeongwoon; Prasad, Vish; Cho, Kyeongjae; Choi, Wonbong

2018-04-01

Among the candidates to replace Li-ion batteries, Li-S cells are an attractive option as their energy density is about five times higher (~2,600 Wh kg -1 ). The success of Li-S cells depends in large part on the utilization of metallic Li as anode material. Metallic lithium, however, is prone to grow parasitic dendrites and is highly reactive to several electrolytes; moreover, Li-S cells with metallic Li are also susceptible to polysulfides dissolution. Here, we show that ~10-nm-thick two-dimensional (2D) MoS 2 can act as a protective layer for Li-metal anodes, greatly improving the performances of Li-S batteries. In particular, we observe stable Li electrodeposition and the suppression of dendrite nucleation sites. The deposition and dissolution process of a symmetric MoS 2 -coated Li-metal cell operates at a current density of 10 mA cm -2 with low voltage hysteresis and a threefold improvement in cycle life compared with using bare Li-metal. In a Li-S full-cell configuration, using the MoS 2 -coated Li as anode and a 3D carbon nanotube-sulfur cathode, we obtain a specific energy density of ~589 Wh kg -1 and a Coulombic efficiency of ~98% for over 1,200 cycles at 0.5 C. Our approach could lead to the realization of high energy density and safe Li-metal-based batteries.

Surveyor Nuclease: a new strategy for a rapid identification of heteroplasmic mitochondrial DNA mutations in patients with respiratory chain defects.

PubMed

Bannwarth, Sylvie; Procaccio, Vincent; Paquis-Flucklinger, Veronique

2005-06-01

Molecular analysis of mitochondrial DNA (mtDNA) is a critical step in diagnosis and genetic counseling of respiratory chain defects. No fast method is currently available for the identification of unknown mtDNA point mutations. We have developed a new strategy based on complete mtDNA PCR amplification followed by digestion with a mismatch-specific DNA endonuclease, Surveyor Nuclease. This enzyme, a member of the CEL nuclease family of plant DNA endonucleases, cleaves double-strand DNA at any mismatch site including base substitutions and small insertions/deletions. After digestion, cleavage products are separated and analyzed by agarose gel electrophoresis. The size of the digestion products indicates the location of the mutation, which is then confirmed and characterized by sequencing. Although this method allows the analysis of 2 kb mtDNA amplicons and the detection of multiple mutations within the same fragment, it does not lead to the identification of homoplasmic base substitutions. Homoplasmic pathogenic mutations have been described. Nevertheless, most homoplasmic base substitutions are neutral polymorphisms while deleterious mutations are typically heteroplasmic. Here, we report that this method can be used to detect mtDNA mutations such as m.3243A>G tRNA(Leu) and m.14709T>C tRNA(Glu) even when they are present at levels as low as 3% in DNA samples derived from patients with respiratory chain defects. Then, we tested five patients suffering from a mitochondrial respiratory chain defect and we identified a variant (m.16189T>C) in two of them, which was previously associated with susceptibility to diabetes and cardiomyopathy. In conclusion, this method can be effectively used to rapidly and completely screen the entire human mitochondrial genome for heteroplasmic mutations and in this context represents an important advance for the diagnosis of mitochondrial diseases.

Protection of Mice against Plasmodium yoelii Sporozoite Challenge with P. yoelii Merozoite Surface Protein 1 DNA Vaccines

PubMed Central

Becker, Sylvia I.; Wang, Ruobing; Hedstrom, Richard C.; Aguiar, Joao C.; Jones, Trevor R.; Hoffman, Stephen L.; Gardner, Malcolm J.

1998-01-01

Immunization of mice with DNA vaccines encoding the full-length form and C and N termini of Plasmodium yoelii merozoite surface protein 1 provided partial protection against sporozoite challenge and resulted in boosting of antibody titers after challenge. In C57BL/6 mice, two DNA vaccines provided protection comparable to that of recombinant protein consisting of the C terminus in Freund’s adjuvant. PMID:9632624

Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells.

PubMed

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

NASA Astrophysics Data System (ADS)

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.

Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia.

PubMed

Rathe, Susan K; Moriarity, Branden S; Stoltenberg, Christopher B; Kurata, Morito; Aumann, Natalie K; Rahrmann, Eric P; Bailey, Natashay J; Melrose, Ellen G; Beckmann, Dominic A; Liska, Chase R; Largaespada, David A

2014-08-13

The evolution from microarrays to transcriptome deep-sequencing (RNA-seq) and from RNA interference to gene knockouts using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and Transcription Activator-Like Effector Nucleases (TALENs) has provided a new experimental partnership for identifying and quantifying the effects of gene changes on drug resistance. Here we describe the results from deep-sequencing of RNA derived from two cytarabine (Ara-C) resistance acute myeloid leukemia (AML) cell lines, and present CRISPR and TALEN based methods for accomplishing complete gene knockout (KO) in AML cells. We found protein modifying loss-of-function mutations in Dck in both Ara-C resistant cell lines. CRISPR and TALEN-based KO of Dck dramatically increased the IC₅₀ of Ara-C and introduction of a DCK overexpression vector into Dck KO clones resulted in a significant increase in Ara-C sensitivity. This effort demonstrates the power of using transcriptome analysis and CRISPR/TALEN-based KOs to identify and verify genes associated with drug resistance.

Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System.

PubMed

Koo, Ok Jae; Park, Sol Ji; Lee, Choongil; Kang, Jung Taek; Kim, Sujin; Moon, Joon Ho; Choi, Ji Yei; Kim, Hyojin; Jang, Goo; Kim, Jin-Soo; Kim, Seokjoong; Lee, Byeong-Chun

2014-03-01

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells (RFP(+)/eGFP(+)) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees.