Sample records for rad-51 filament disassembly

  1. Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament

    PubMed Central

    Ma, Chu Jian; Gibb, Bryan; Kwon, YoungHo; Sung, Patrick; Greene, Eric C.

    2017-01-01

    Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly. PMID:27903895

  2. Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament.

    PubMed

    Ma, Chu Jian; Gibb, Bryan; Kwon, YoungHo; Sung, Patrick; Greene, Eric C

    2017-01-25

    Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Srs2 prevents Rad51 filament formation by repetitive motion on DNA.

    PubMed

    Qiu, Yupeng; Antony, Edwin; Doganay, Sultan; Koh, Hye Ran; Lohman, Timothy M; Myong, Sua

    2013-01-01

    Srs2 dismantles presynaptic Rad51 filaments and prevents its re-formation as an anti-recombinase. However, the molecular mechanism by which Srs2 accomplishes these tasks remains unclear. Here we report a single-molecule fluorescence study of the dynamics of Rad51 filament formation and its disruption by Srs2. Rad51 forms filaments on single-stranded DNA by sequential binding of primarily monomers and dimers in a 5'-3' direction. One Rad51 molecule binds to three nucleotides, and six monomers are required to achieve a stable nucleation cluster. Srs2 exhibits ATP-dependent repetitive motion on single-stranded DNA and this activity prevents re-formation of the Rad51 filament. The same activity of Srs2 cannot prevent RecA filament formation, indicating its specificity for Rad51. Srs2's DNA-unwinding activity is greatly suppressed when Rad51 filaments form on duplex DNA. Taken together, our results reveal an exquisite and highly specific mechanism by which Srs2 regulates the Rad51 filament formation.

  4. Single-molecule localization microscopy reveals molecular transactions during RAD51 filament assembly at cellular DNA damage sites

    PubMed Central

    Haas, Kalina T; Lee, MiYoung; Esposito, Alessandro; Venkitaraman, Ashok R

    2018-01-01

    Abstract RAD51 recombinase assembles on single-stranded (ss)DNA substrates exposed by DNA end-resection to initiate homologous recombination (HR), a process fundamental to genome integrity. RAD51 assembly has been characterized using purified proteins, but its ultrastructural topography in the cell nucleus is unexplored. Here, we combine cell genetics with single-molecule localization microscopy and a palette of bespoke analytical tools, to visualize molecular transactions during RAD51 assembly in the cellular milieu at resolutions approaching 30–40 nm. In several human cell types, RAD51 focalizes in clusters that progressively extend into long filaments, which abut—but do not overlap—with globular bundles of replication protein A (RPA). Extended filaments alter topographically over time, suggestive of succeeding steps in HR. In cells depleted of the tumor suppressor protein BRCA2, or overexpressing its RAD51-binding BRC repeats, RAD51 fails to assemble at damage sites, although RPA accumulates unhindered. By contrast, in cells lacking a BRCA2 carboxyl (C)-terminal region targeted by cancer-causing mutations, damage-induced RAD51 assemblies initiate but do not extend into filaments. We suggest a model wherein RAD51 assembly proceeds concurrently with end-resection at adjacent sites, via an initiation step dependent on the BRC repeats, followed by filament extension through the C-terminal region of BRCA2. PMID:29309696

  5. 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

  6. Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

    PubMed

    Fornander, Louise H; Frykholm, Karolin; Reymer, Anna; Renodon-Cornière, Axelle; Takahashi, Masayuki; Nordén, Bengt

    2012-06-01

    Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

  7. Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

    PubMed Central

    Tsabar, Michael; Mason, Jennifer M.; Chan, Yuen-Ling; Bishop, Douglas K.; Haber, James E.

    2015-01-01

    Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments. PMID:26019181

  8. Promotion of Homologous Recombination and Genomic Stability byRAD51AP1 via RAD51 Recombinase Enhancement

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

    Wiese, Claudia; Dray, Eloise; Groesser, Torsten

    2007-04-11

    Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand pairing step in HR. RAD51AP1 (RAD51 Associated Protein 1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA damaging treatment. Purified RAD51AP1 binds dsDNA and RAD51, and it greatly stimulates the RAD51-mediated D-loop reaction. Biochemical and cytological results show that RAD51AP1more » functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide the first evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.« less

  9. Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament

    PubMed Central

    Fornander, Louise H.; Renodon-Cornière, Axelle; Kuwabara, Naoyuki; Ito, Kentaro; Tsutsui, Yasuhiro; Shimizu, Toshiyuki; Iwasaki, Hiroshi; Nordén, Bengt; Takahashi, Masayuki

    2014-01-01

    The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction. PMID:24304898

  10. Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament.

    PubMed

    Fornander, Louise H; Renodon-Cornière, Axelle; Kuwabara, Naoyuki; Ito, Kentaro; Tsutsui, Yasuhiro; Shimizu, Toshiyuki; Iwasaki, Hiroshi; Nordén, Bengt; Takahashi, Masayuki

    2014-02-01

    The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.

  11. Three New Structures of Left-Handed RadA Helical Filaments: Structural Flexibility of N-Terminal Domain Is Critical for Recombinase Activity

    PubMed Central

    Chang, Yu-Wei; Ko, Tzu-Ping; Lee, Chien-Der; Chang, Yuan-Chih; Lin, Kuei-Ann; Chang, Chia-Seng; Wang, Andrew H.-J.; Wang, Ting-Fang

    2009-01-01

    RecA family proteins, including bacterial RecA, archaeal RadA, and eukaryotic Dmc1 and Rad51, mediate homologous recombination, a reaction essential for maintaining genome integrity. In the presence of ATP, these proteins bind a single-strand DNA to form a right-handed nucleoprotein filament, which catalyzes pairing and strand exchange with a homologous double-stranded DNA (dsDNA), by as-yet unknown mechanisms. We recently reported a structure of RadA left-handed helical filament, and here present three new structures of RadA left-handed helical filaments. Comparative structural analysis between different RadA/Rad51 helical filaments reveals that the N-terminal domain (NTD) of RadA/Rad51, implicated in dsDNA binding, is highly flexible. We identify a hinge region between NTD and polymerization motif as responsible for rigid body movement of NTD. Mutant analysis further confirms that structural flexibility of NTD is essential for RadA's recombinase activity. These results support our previous hypothesis that ATP-dependent axial rotation of RadA nucleoprotein helical filament promotes homologous recombination. PMID:19295907

  12. Rad51 is an accessory factor for Dmc1-mediated joint molecule formation during meiosis.

    PubMed

    Cloud, Veronica; Chan, Yuen-Ling; Grubb, Jennifer; Budke, Brian; Bishop, Douglas K

    2012-09-07

    Meiotic recombination in budding yeast requires two RecA-related proteins, Rad51 and Dmc1, both of which form filaments on DNA capable of directing homology search and catalyzing formation of homologous joint molecules (JMs) and strand exchange. With use of a separation-of-function mutant form of Rad51 that retains filament-forming but not JM-forming activity, we show that the JM activity of Rad51 is fully dispensable for meiotic recombination. The corresponding mutation in Dmc1 causes a profound recombination defect, demonstrating Dmc1's JM activity alone is responsible for meiotic recombination. We further provide biochemical evidence that Rad51 acts with Mei5-Sae3 as a Dmc1 accessory factor. Thus, Rad51 is a multifunctional protein that catalyzes recombination directly in mitosis and indirectly, via Dmc1, during meiosis.

  13. Cellular Dynamics of Rad51 and Rad54 in Response to Postreplicative Stress and DNA Damage in HeLa Cells.

    PubMed

    Choi, Eui-Hwan; Yoon, Seobin; Hahn, Yoonsoo; Kim, Keun P

    2017-02-01

    Homologous recombination (HR) is necessary for maintenance of genomic integrity and prevention of various mutations in tumor suppressor genes and proto-oncogenes. Rad51 and Rad54 are key HR factors that cope with replication stress and DNA breaks in eukaryotes. Rad51 binds to single-stranded DNA (ssDNA) to form the presynaptic filament that promotes a homology search and DNA strand exchange, and Rad54 stimulates the strand-pairing function of Rad51. Here, we studied the molecular dynamics of Rad51 and Rad54 during the cell cycle of HeLa cells. These cells constitutively express Rad51 and Rad54 throughout the entire cell cycle, and the formation of foci immediately increased in response to various types of DNA damage and replication stress, except for caffeine, which suppressed the Rad51-dependent HR pathway. Depletion of Rad51 caused severe defects in response to postreplicative stress. Accordingly, HeLa cells were arrested at the G2-M transition although a small amount of Rad51 was steadily maintained in HeLa cells. Our results suggest that cell cycle progression and proliferation of HeLa cells can be tightly controlled by the abundance of HR proteins, which are essential for the rapid response to postreplicative stress and DNA damage stress.

  14. Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells

    NASA Technical Reports Server (NTRS)

    Wiese, Claudia; Collins, David W.; Albala, Joanna S.; Thompson, Larry H.; Kronenberg, Amy; Schild, David; Chatterjee, A. (Principal Investigator)

    2002-01-01

    Homologous recombinational repair of DNA double-strand breaks and crosslinks in human cells is likely to require Rad51 and the five Rad51 paralogs (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2 and Rad51D/Rad51L3), as has been shown in chicken and rodent cells. Previously, we reported on the interactions among these proteins using baculovirus and two- and three-hybrid yeast systems. To test for interactions involving XRCC3 and Rad51C, stable human cell lines have been isolated that express (His)6-tagged versions of XRCC3 or Rad51C. Ni2+-binding experiments demonstrate that XRCC3 and Rad51C interact in human cells. In addition, we find that Rad51C, but not XRCC3, interacts directly or indirectly with Rad51B, Rad51D and XRCC2. These results argue that there are at least two complexes of Rad51 paralogs in human cells (Rad51C-XRCC3 and Rad51B-Rad51C-Rad51D-XRCC2), both containing Rad51C. Moreover, Rad51 is not found in these complexes. X-ray treatment did not alter either the level of any Rad51 paralog or the observed interactions between paralogs. However, the endogenous level of Rad51C is moderately elevated in the XRCC3-overexpressing cell line, suggesting that dimerization between these proteins might help stabilize Rad51C.

  15. Actin-filament disassembly: it takes two to shrink them fast.

    PubMed

    Winterhoff, Moritz; Faix, Jan

    2015-06-01

    Actin-filament disassembly is indispensable for replenishing the pool of polymerizable actin and allows continuous dynamic remodelling of the actin cytoskeleton. A new study now reveals that ADF/cofilin preferentially dismantles branched networks and provides new insights into the collaborative work of ADF/cofilin and Aip1 on filament disassembly at the molecular level. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Contribution of Germline Mutations in the RAD51B, RAD51C, and RAD51D Genes to Ovarian Cancer in the Population

    PubMed Central

    Song, Honglin; Dicks, Ed; Ramus, Susan J.; Tyrer, Jonathan P.; Intermaggio, Maria P.; Hayward, Jane; Edlund, Christopher K.; Conti, David; Harrington, Patricia; Fraser, Lindsay; Philpott, Susan; Anderson, Christopher; Rosenthal, Adam; Gentry-Maharaj, Aleksandra; Bowtell, David D.; Alsop, Kathryn; Cicek, Mine S.; Cunningham, Julie M.; Fridley, Brooke L.; Alsop, Jennifer; Jimenez-Linan, Mercedes; Høgdall, Estrid; Høgdall, Claus K.; Jensen, Allan; Kjaer, Susanne Krüger; Lubiński, Jan; Huzarski, Tomasz; Jakubowska, Anna; Gronwald, Jacek; Poblete, Samantha; Lele, Shashi; Sucheston-Campbell, Lara; Moysich, Kirsten B.; Odunsi, Kunle; Goode, Ellen L.; Menon, Usha; Jacobs, Ian J.; Gayther, Simon A.; Pharoah, Paul D.P.

    2015-01-01

    Purpose The aim of this study was to estimate the contribution of deleterious mutations in the RAD51B, RAD51C, and RAD51D genes to invasive epithelial ovarian cancer (EOC) in the population and in a screening trial of individuals at high risk of ovarian cancer. Patients and Methods The coding sequence and splice site boundaries of the three RAD51 genes were sequenced and analyzed in germline DNA from a case-control study of 3,429 patients with invasive EOC and 2,772 controls as well as in 2,000 unaffected women who were BRCA1/BRCA2 negative from the United Kingdom Familial Ovarian Cancer Screening Study (UK_FOCSS) after quality-control analysis. Results In the case-control study, we identified predicted deleterious mutations in 28 EOC cases (0.82%) compared with three controls (0.11%; P < .001). Mutations in EOC cases were more frequent in RAD51C (14 occurrences, 0.41%) and RAD51D (12 occurrences, 0.35%) than in RAD51B (two occurrences, 0.06%). RAD51C mutations were associated with an odds ratio of 5.2 (95% CI, 1.1 to 24; P = .035), and RAD51D mutations conferred an odds ratio of 12 (95% CI, 1.5 to 90; P = .019). We identified 13 RAD51 mutations (0.65%) in unaffected UK_FOCSS participants (RAD51C, n = 7; RAD51D, n = 5; and RAD51B, n = 1), which was a significantly greater rate than in controls (P < .001); furthermore, RAD51 mutation carriers were more likely than noncarriers to have a family history of ovarian cancer (P < .001). Conclusion These results confirm that RAD51C and RAD51D are moderate ovarian cancer susceptibility genes and suggest that they confer levels of risk of EOC that may warrant their use alongside BRCA1 and BRCA2 in routine clinical genetic testing. PMID:26261251

  17. Molecular Basis for Enhancement of the Meiotic DMCI Recombinase by RAD51AP1

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

    Dray, Eloise; Dunlop, Myun Hwa; Kauppi, Liisa

    Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 Associated Protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex DNA partnermore » and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional co-operation is dependent on complex formation between DMC1 and RAD51AP1, and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci co-localize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.« less

  18. Dmc1 catalyzes interhomolog joint molecule formation in meiosis with Rad51 and Mei5-Sae3 as accessory factors

    PubMed Central

    Cloud, Veronica; Chan, Yuen-Ling; Grubb, Jennifer; Budke, Brian; Bishop, Douglas K.

    2014-01-01

    Meiotic recombination in budding yeast requires two RecA-related proteins, Rad51 and Dmc1, both of which form filaments on DNA capable of directing homology search and catalyzing formation of homologous joint molecules (JMs) and strand exchange. Using a separation-of-function mutant form of Rad51, that retains filament-forming but not JM forming activity, we show that the JM activity of Rad51 is fully dispensable for meiotic recombination. The corresponding mutation in Dmc1 causes a profound recombination defect, demonstrating Dmc1’s JM activity alone is responsible for meiotic recombination. We further provide biochemical evidence that Rad51 acts with Mei5-Sae3 as a Dmc1 accessory factor. Thus, Rad51 is a multifunctional protein that catalyzes recombination directly in mitosis and indirectly, via Dmc1, during meiosis. PMID:22955832

  19. Cellular redistribution of Rad51 in response to DNA damage: novel role for Rad51C.

    PubMed

    Gildemeister, Otto S; Sage, Jay M; Knight, Kendall L

    2009-11-13

    Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51 may contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damage-induced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damage-induced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.

  20. Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro

    NASA Technical Reports Server (NTRS)

    Lio, Yi-Ching; Mazin, Alexander V.; Kowalczykowski, Stephen C.; Chen, David J.

    2003-01-01

    The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)(6), and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B.Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.

  1. Small Rad51 and Dmc1 Complexes Often Co-occupy Both Ends of a Meiotic DNA Double Strand Break

    PubMed Central

    Brown, M. Scott; Grubb, Jennifer; Zhang, Annie; Rust, Michael J.; Bishop, Douglas K.

    2015-01-01

    The Eukaryotic RecA-like proteins Rad51 and Dmc1 cooperate during meiosis to promote recombination between homologous chromosomes by repairing programmed DNA double strand breaks (DSBs). Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci. Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm. Paired co-foci remain prevalent when DSBs are dramatically reduced or when strand exchange or synapsis is blocked. Super-resolution dSTORM microscopy reveals that individual foci observed by conventional light microscopy are often composed of two or more substructures. The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt) Rad51 filaments and also by one or more short Dmc1 filaments. PMID:26719980

  2. Domain mapping of the Rad51 paralog protein complexes

    PubMed Central

    Miller, Kristi A.; Sawicka, Dorota; Barsky, Daniel; Albala, Joanna S.

    2004-01-01

    The five human Rad51 paralogs are suggested to play an important role in the maintenance of genome stability through their function in DNA double-strand break repair. These proteins have been found to form two distinct complexes in vivo, Rad51B–Rad51C–Rad51D–Xrcc2 (BCDX2) and Rad51C–Xrcc3 (CX3). Based on the recent Pyrococcus furiosus Rad51 structure, we have used homology modeling to design deletion mutants of the Rad51 paralogs. The models of the human Rad51B, Rad51C, Xrcc3 and murine Rad51D (mRad51D) proteins reveal distinct N-terminal and C-terminal domains connected by a linker region. Using yeast two-hybrid and co-immunoprecipitation techniques, we have demonstrated that a fragment of Rad51B containing amino acid residues 1–75 interacts with the C-terminus and linker of Rad51C, residues 79–376, and this region of Rad51C also interacts with mRad51D and Xrcc3. We have also determined that the N-terminal domain of mRad51D, residues 4–77, binds to Xrcc2 while the C-terminal domain of mRad51D, residues 77–328, binds Rad51C. By this, we have identified the binding domains of the BCDX2 and CX3 complexes to further characterize the interaction of these proteins and propose a scheme for the three-dimensional architecture of the BCDX2 and CX3 paralog complexes. PMID:14704354

  3. The N-terminal DNA-binding domain of Rad52 promotes RAD51-independent recombination in Saccharomyces cerevisiae.

    PubMed Central

    Tsukamoto, Mariko; Yamashita, Kentaro; Miyazaki, Toshiko; Shinohara, Miki; Shinohara, Akira

    2003-01-01

    In Saccharomyces cerevisiae, the Rad52 protein plays a role in both RAD51-dependent and RAD51-independent recombination pathways. We characterized a rad52 mutant, rad52-329, which lacks the C-terminal Rad51-interacting domain, and studied its role in RAD51-independent recombination. The rad52-329 mutant is completely defective in mating-type switching, but partially proficient in recombination between inverted repeats. We also analyzed the effect of the rad52-329 mutant on telomere recombination. Yeast cells lacking telomerase maintain telomere length by recombination. The rad52-329 mutant is deficient in RAD51-dependent telomere recombination, but is proficient in RAD51-independent telomere recombination. In addition, we examined the roles of other recombination genes in the telomere recombination. The RAD51-independent recombination in the rad52-329 mutant is promoted by a paralogue of Rad52, Rad59. All components of the Rad50-Mre11-Xrs2 complex are also important, but not essential, for RAD51-independent telomere recombination. Interestingly, RAD51 inhibits the RAD51-independent, RAD52-dependent telomere recombination. These findings indicate that Rad52 itself, and more precisely its N-terminal DNA-binding domain, promote an essential reaction in recombination in the absence of RAD51. PMID:14704160

  4. Distinct RAD51 Associations with RAD52 and BCCIP in Response to DNA Damage and Replication Stress

    PubMed Central

    Wray, Justin; Liu, Jingmei; Nickoloff, Jac A.; Shen, Zhiyuan

    2009-01-01

    RAD51 has critical roles in homologous recombination (HR) repair of DNA double-strand breaks (DSB) and restarting stalled or collapsed replication forks. In yeast, Rad51 function is facilitated by Rad52 and other “mediators.” Mammalian cells express RAD52, but BRCA2 may have supplanted RAD52 in mediating RAD51 loading onto ssDNA. BCCIP interacts with BRCA2, and both proteins are important for RAD51 focus formation after ionizing radiation and HR repair of DSBs. Nonetheless, mammalian RAD52 shares biochemical activities with yeast Rad52, including RAD51 binding and single-strand annealing, suggesting a conserved role in HR. Because RAD52 and RAD51 associate, and RAD51 and BCCIP associate, we investigated the colocalization of RAD51 with BCCIP and RAD52 in human cells. We found that RAD51 colocalizes with BCCIP early after ionizing radiation, with RAD52 later, and there was little colocalization of BCCIP and RAD52. RAD52 foci are induced to a greater extent by hydroxyurea, which stalls replication forks, than by ionizing radiation. Using fluorescence recovery after photo bleaching, we show that RAD52 mobility is reduced to a greater extent by hydroxyurea than ionizing radiation. However, BCCIP showed no changes in mobility after hydroxyurea or ionizing radiation. We propose that BCCIP-dependent repair of DSBs by HR is an early RAD51 response to ionizing radiation–induced DNA damage, and that RAD52-dependent HR occurs later to restart a subset of blocked or collapsed replication forks. RAD52 and BRCA2 seem to act in parallel pathways, suggesting that targeting RAD52 in BRCA2-deficient tumors may be effective in treating these tumors. PMID:18413737

  5. Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner.

    PubMed

    Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

    2016-03-04

    Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Dbl2 Regulates Rad51 and DNA Joint Molecule Metabolism to Ensure Proper Meiotic Chromosome Segregation

    PubMed Central

    Hyppa, Randy W.; Benko, Zsigmond; Misova, Ivana; Schleiffer, Alexander; Smith, Gerald R.; Gregan, Juraj

    2016-01-01

    To identify new proteins required for faithful meiotic chromosome segregation, we screened a Schizosaccharomyces pombe deletion mutant library and found that deletion of the dbl2 gene led to missegregation of chromosomes during meiosis. Analyses of both live and fixed cells showed that dbl2Δ mutant cells frequently failed to segregate homologous chromosomes to opposite poles during meiosis I. Removing Rec12 (Spo11 homolog) to eliminate meiotic DNA double-strand breaks (DSBs) suppressed the segregation defect in dbl2Δ cells, indicating that Dbl2 acts after the initiation of meiotic recombination. Analyses of DSBs and Holliday junctions revealed no significant defect in their formation or processing in dbl2Δ mutant cells, although some Rec12-dependent DNA joint molecules persisted late in meiosis. Failure to segregate chromosomes in the absence of Dbl2 correlated with persistent Rad51 foci, and deletion of rad51 or genes encoding Rad51 mediators also suppressed the segregation defect of dbl2Δ. Formation of foci of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments, was impaired in dbl2Δ cells. Our results suggest that Dbl2 is a novel regulator of Fbh1 and thereby Rad51-dependent DSB repair required for proper meiotic chromosome segregation and viable sex cell formation. The wide conservation of these proteins suggests that our results apply to many species. PMID:27304859

  7. Differential RPA-1 and RAD-51 recruitment in vivo throughout the C. elegans germline, as revealed by laser microirradiation

    PubMed Central

    Koury, Emily; Harrell, Kailey

    2018-01-01

    Abstract Studies of the repair pathways associated with DNA double strand breaks (DSBs) are numerous, and provide evidence for cell-cycle specific regulation of homologous recombination (HR) by the regulation of its associated proteins. Laser microirradiation is a well-established method to examine in vitro kinetics of repair and allows for live-imaging of DSB repair from the moment of induction. Here we apply this method to whole, live organisms, introducing an effective system to analyze exogenous, microirradiation-induced breaks in the Caenorhabditis elegans germline. Through this method we observed the sequential kinetics of the recruitment of ssDNA binding proteins RPA-1 and RAD-51 in vivo. We analyze these kinetics throughout different regions of the germline, and thus throughout a range of developmental stages of mitotic and meiotic nuclei. Our analysis demonstrates a largely conserved timing of recruitment of ssDNA binding proteins to DSBs throughout the germline, with a delay of RAD-51 recruitment at mid-pachytene nuclei. Microirradiated nuclei are viable and undergo a slow kinetics of resolution. We observe RPA-1 and RAD-51 colocalization for hours post-microirradiation throughout the germline, suggesting that there are mixed RPA-1/RAD-51 filaments. Finally, through live imaging analysis we observed RAD-51 foci movement with low frequency of coalescence. PMID:29244155

  8. Differential RPA-1 and RAD-51 recruitment in vivo throughout the C. elegans germline, as revealed by laser microirradiation.

    PubMed

    Koury, Emily; Harrell, Kailey; Smolikove, Sarit

    2018-01-25

    Studies of the repair pathways associated with DNA double strand breaks (DSBs) are numerous, and provide evidence for cell-cycle specific regulation of homologous recombination (HR) by the regulation of its associated proteins. Laser microirradiation is a well-established method to examine in vitro kinetics of repair and allows for live-imaging of DSB repair from the moment of induction. Here we apply this method to whole, live organisms, introducing an effective system to analyze exogenous, microirradiation-induced breaks in the Caenorhabditis elegans germline. Through this method we observed the sequential kinetics of the recruitment of ssDNA binding proteins RPA-1 and RAD-51 in vivo. We analyze these kinetics throughout different regions of the germline, and thus throughout a range of developmental stages of mitotic and meiotic nuclei. Our analysis demonstrates a largely conserved timing of recruitment of ssDNA binding proteins to DSBs throughout the germline, with a delay of RAD-51 recruitment at mid-pachytene nuclei. Microirradiated nuclei are viable and undergo a slow kinetics of resolution. We observe RPA-1 and RAD-51 colocalization for hours post-microirradiation throughout the germline, suggesting that there are mixed RPA-1/RAD-51 filaments. Finally, through live imaging analysis we observed RAD-51 foci movement with low frequency of coalescence. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. RAD51B in Familial Breast Cancer

    PubMed Central

    Pelttari, Liisa M.; Khan, Sofia; Vuorela, Mikko; Kiiski, Johanna I.; Vilske, Sara; Nevanlinna, Viivi; Ranta, Salla; Schleutker, Johanna; Winqvist, Robert; Kallioniemi, Anne; Dörk, Thilo; Bogdanova, Natalia V.; Figueroa, Jonine; Pharoah, Paul D. P.; Schmidt, Marjanka K.; Dunning, Alison M.; García-Closas, Montserrat; Bolla, Manjeet K.; Dennis, Joe; Michailidou, Kyriaki; Wang, Qin; Hopper, John L.; Southey, Melissa C.; Rosenberg, Efraim H.; Fasching, Peter A.; Beckmann, Matthias W.; Peto, Julian; dos-Santos-Silva, Isabel; Sawyer, Elinor J.; Tomlinson, Ian; Burwinkel, Barbara; Surowy, Harald; Guénel, Pascal; Truong, Thérèse; Bojesen, Stig E.; Nordestgaard, Børge G.; Benitez, Javier; González-Neira, Anna; Neuhausen, Susan L.; Anton-Culver, Hoda; Brenner, Hermann; Arndt, Volker; Meindl, Alfons; Schmutzler, Rita K.; Brauch, Hiltrud; Brüning, Thomas; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Hartikainen, Jaana M.; Chenevix-Trench, Georgia; Van Dyck, Laurien; Janssen, Hilde; Chang-Claude, Jenny; Rudolph, Anja; Radice, Paolo; Peterlongo, Paolo; Hallberg, Emily; Olson, Janet E.; Giles, Graham G.; Milne, Roger L.; Haiman, Christopher A.; Schumacher, Fredrick; Simard, Jacques; Dumont, Martine; Kristensen, Vessela; Borresen-Dale, Anne-Lise; Zheng, Wei; Beeghly-Fadiel, Alicia; Grip, Mervi; Andrulis, Irene L.; Glendon, Gord; Devilee, Peter; Seynaeve, Caroline; Hooning, Maartje J.; Collée, Margriet; Cox, Angela; Cross, Simon S.; Shah, Mitul; Luben, Robert N.; Hamann, Ute; Torres, Diana; Jakubowska, Anna; Lubinski, Jan; Couch, Fergus J.; Yannoukakos, Drakoulis; Orr, Nick; Swerdlow, Anthony; Darabi, Hatef; Li, Jingmei; Czene, Kamila; Hall, Per; Easton, Douglas F.; Mattson, Johanna; Blomqvist, Carl; Aittomäki, Kristiina; Nevanlinna, Heli

    2016-01-01

    Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737 and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer predisposition, particularly in the context of familial breast cancer in Finland. We sequenced the coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for identification of possible recurrent founder mutations. In addition, we studied the known rs999737, rs2588809, and rs1314913 SNPs and RAD51B haplotypes in 44,791 breast cancer cases and 43,583 controls from 40 studies participating in the Breast Cancer Association Consortium (BCAC) that were genotyped on a custom chip (iCOGS). We identified one putatively pathogenic missense mutation c.541C>T among the Finnish cancer patients and subsequently genotyped the mutation in additional breast cancer cases (n = 5259) and population controls (n = 3586) from Finland and Belarus. No significant association with breast cancer risk was seen in the meta-analysis of the Finnish datasets or in the large BCAC dataset. The association with previously identified risk variants rs999737, rs2588809, and rs1314913 was replicated among all breast cancer cases and also among familial cases in the BCAC dataset. The most significant association was observed for the haplotype carrying the risk-alleles of all the three SNPs both among all cases (odds ratio (OR): 1.15, 95% confidence interval (CI): 1.11–1.19, P = 8.88 x 10−16) and among familial cases (OR: 1.24, 95% CI: 1.16–1.32, P = 6.19 x 10−11), compared to the haplotype with the respective protective alleles. Our results suggest that loss-of-function mutations in RAD51B are rare, but common variation at the RAD51B region is significantly associated with familial breast cancer risk. PMID:27149063

  10. RAD51 interconnects between DNA replication, DNA repair and immunity.

    PubMed

    Bhattacharya, Souparno; Srinivasan, Kalayarasan; Abdisalaam, Salim; Su, Fengtao; Raj, Prithvi; Dozmorov, Igor; Mishra, Ritu; Wakeland, Edward K; Ghose, Subroto; Mukherjee, Shibani; Asaithamby, Aroumougame

    2017-05-05

    RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation

    PubMed Central

    Dobson, Rachel; Stockdale, Christopher; Lapsley, Craig; Wilkes, Jonathan; McCulloch, Richard

    2011-01-01

    Homologous recombination in Trypanosoma brucei is used for moving variant surface glycoprotein (VSG) genes into expression sites during immune evasion by antigenic variation. A major route for such VSG switching is gene conversion reactions in which RAD51, a universally conserved recombinase, catalyses homology-directed strand exchange. In any eukaryote, RAD51-directed strand exchange in vivo is mediated by further factors, including RAD51-related proteins termed Rad51 paralogues. These appear to be ubiquitously conserved, although their detailed roles in recombination remain unclear. In T. brucei, four putative RAD51 paralogue genes have been identified by sequence homology. Here we show that all four RAD51 paralogues act in DNA repair, recombination and RAD51 subnuclear dynamics, though not equivalently, while mutation of only one RAD51 paralogue gene significantly impedes VSG switching. We also show that the T. brucei RAD51 paralogues interact, and that the complexes they form may explain the distinct phenotypes of the mutants as well as observed expression interdependency. Finally, we document the Rad51 paralogues that are encoded by a wide range of protists, demonstrating that the Rad51 paralogue repertoire in T. brucei is unusually large among microbial eukaryotes and that one member of the protein family corresponds with a key, conserved eukaryotic Rad51 paralogue. PMID:21615552

  12. Prolonged exposure to particulate chromate inhibits RAD51 nuclear import mediator proteins.

    PubMed

    Browning, Cynthia L; Wise, John Pierce

    2017-09-15

    Particulate hexavalent chromium (Cr(VI)) is a human lung carcinogen and a human health concern. The induction of structural chromosome instability is considered to be a driving mechanism of Cr(VI)-induced carcinogenesis. Homologous recombination repair protects against Cr(VI)-induced chromosome damage, due to its highly accurate repair of Cr(VI)-induced DNA double strand breaks. However, recent studies demonstrate Cr(VI) inhibits homologous recombination repair through the misregulation of RAD51. RAD51 is an essential protein in HR repair that facilitates the search for a homologous sequence. Recent studies show prolonged Cr(VI) exposure prevents proper RAD51 subcellular localization, causing it to accumulate in the cytoplasm. Since nuclear import of RAD51 is crucial to its function, this study investigated the effect of Cr(VI) on the RAD51 nuclear import mediators, RAD51C and BRCA2. We show acute (24h) Cr(VI) exposure induces the proper localization of RAD51C and BRCA2. In contrast, prolonged (120h) exposure increased the cytoplasmic localization of both proteins, although RAD51C localization was more severely impaired. These results correlate temporally with the previously reported Cr(VI)-induced RAD51 cytoplasmic accumulation. In addition, we found Cr(VI) does not inhibit interaction between RAD51 and its nuclear import mediators. Altogether, our results suggest prolonged Cr(VI) exposure inhibits the nuclear import of RAD51C, and to a lesser extent, BRCA2, which results in the cytoplasmic accumulation of RAD51. Cr(VI)-induced inhibition of nuclear import may play a key role in its carcinogenic mechanism since the nuclear import of many tumor suppressor proteins and DNA repair proteins is crucial to their function. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Three-dimensional microscopy of the Rad51 recombination protein during meiotic prophase.

    PubMed Central

    Franklin, A E; McElver, J; Sunjevaric, I; Rothstein, R; Bowen, B; Cande, W Z

    1999-01-01

    An open question in meiosis is whether the Rad51 recombination protein functions solely in meiotic recombination or whether it is also involved in the chromosome homology search. To address this question, we have performed three-dimensional high-resolution immunofluorescence microscopy to visualize native Rad51 structures in maize male meiocytes. Maize has two closely related RAD51 genes that are expressed at low levels in differentiated tissues and at higher levels in mitotic and meiotic tissues. Cells and nuclei were specially fixed and embedded in polyacrylamide to maintain both native chromosome structure and the three dimensionality of the specimens. Analysis of Rad51 in maize meiocytes revealed that when chromosomes condense during leptotene, Rad51 is diffuse within the nucleus. Rad51 foci form on the chromosomes at the beginning of zygotene and rise to approximately 500 per nucleus by mid-zygotene when chromosomes are pairing and synapsing. During chromosome pairing, we consistently found two contiguous Rad51 foci on paired chromosomes. These paired foci may identify the sites where DNA sequence homology is being compared. During pachytene, the number of Rad51 foci drops to seven to 22 per nucleus. This higher number corresponds approximately to the number of chiasmata in maize meiosis. These observations are consistent with a role for Rad51 in the homology search phase of chromosome pairing in addition to its known role in meiotic recombination. PMID:10330467

  14. Enhancement of the RAD51 Recombinase Activity by the Tumor Suppressor PALB2

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

    Dray, Eloise; Etchin, Julia; Wiese, Claudia

    2010-08-24

    Homologous recombination mediated by the RAD51 recombinase helps eliminate chromosomal lesions, such as DNA double-stranded breaks induced by radiation or arising from injured DNA replication forks. The tumor suppressors BRCA2 and PALB2 act together to deliver RAD51 to chromosomal lesions to initiate repair. Here we document a new function of PALB2 in the enhancement of RAD51's ability to form the D-loop. We show that PALB2 binds DNA and physically interacts with RAD51. Importantly, while PALB2 alone stimulates D-loop formation, a cooperative effect is seen with RAD51AP1, an enhancer of RAD51. This stimulation stems from PALB2's ability to function with RAD51more » and RAD51AP1 to assemble the synaptic complex. Our results help unveil a multi-faceted role of PALB2 in chromosome damage repair. Since PALB2 mutations can cause breast and other tumors or lead to Fanconi anemia, our findings are important for understanding the mechanism of tumor suppression in humans.« less

  15. Characterisation of the novel deleterious RAD51C p.Arg312Trp variant and prioritisation criteria for functional analysis of RAD51C missense changes.

    PubMed

    Gayarre, Javier; Martín-Gimeno, Paloma; Osorio, Ana; Paumard, Beatriz; Barroso, Alicia; Fernández, Victoria; de la Hoya, Miguel; Rojo, Alejandro; Caldés, Trinidad; Palacios, José; Urioste, Miguel; Benítez, Javier; García, María J

    2017-09-26

    Despite a high prevalence of deleterious missense variants, most studies of RAD51C ovarian cancer susceptibility gene only provide in silico pathogenicity predictions of missense changes. We identified a novel deleterious RAD51C missense variant (p.Arg312Trp) in a high-risk family, and propose a criteria to prioritise RAD51C missense changes qualifying for functional analysis. To evaluate pathogenicity of p.Arg312Trp variant we used sequence homology, loss of heterozygosity (LOH) and segregation analysis, and a comprehensive functional characterisation. To define a functional-analysis prioritisation criteria, we used outputs for the known functionally confirmed deleterious and benign RAD51C missense changes from nine pathogenicity prediction algorithms. The p.Arg312Trp variant failed to correct mitomycin and olaparib hypersensitivity and to complement abnormal RAD51C foci formation according to functional assays, which altogether with LOH and segregation data demonstrated deleteriousness. Prioritisation criteria were based on the number of predictors providing a deleterious output, with a minimum of 5 to qualify for testing and a PredictProtein score greater than 33 to assign high-priority indication. Our study points to a non-negligible number of RAD51C missense variants likely to impair protein function, provides a guideline to prioritise and encourage their selection for functional analysis and anticipates that reference laboratories should have available resources to conduct such assays.

  16. RAD51 Is a Selective DNA Repair Target to Radiosensitize Glioma Stem Cells.

    PubMed

    King, Harry O; Brend, Tim; Payne, Helen L; Wright, Alexander; Ward, Thomas A; Patel, Karan; Egnuni, Teklu; Stead, Lucy F; Patel, Anjana; Wurdak, Heiko; Short, Susan C

    2017-01-10

    Patients with glioblastoma die from local relapse despite surgery and high-dose radiotherapy. Resistance to radiotherapy is thought to be due to efficient DNA double-strand break (DSB) repair in stem-like cells able to survive DNA damage and repopulate the tumor. We used clinical samples and patient-derived glioblastoma stem cells (GSCs) to confirm that the DSB repair protein RAD51 is highly expressed in GSCs, which are reliant on RAD51-dependent DSB repair after radiation. RAD51 expression and RAD51 foci numbers fall when these cells move toward astrocytic differentiation. In GSCs, the small-molecule RAD51 inhibitors RI-1 and B02 prevent RAD51 focus formation, reduce DNA DSB repair, and cause significant radiosensitization. We further demonstrate that treatment with these agents combined with radiation promotes loss of stem cells defined by SOX2 expression. This indicates that RAD51-dependent repair represents an effective and specific target in GSCs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Location of RAD51-like protein during meiotic prophase in Eimeria tenella.

    PubMed

    Del Cacho, Emilio; Gallego, Margarita; Pagés, Marc; Barbero, José Luís; Monteagudo, Luís; Sánchez-Acedo, Caridad

    2011-05-31

    This study focuses on reporting events in Eimeria tenella oocysts from early to late prophase I in terms of RAD51 protein in association with the synaptonemal complex formed between homologous chromosomes. The aim of the study was the sequential localization of RAD51 protein, which is involved in the repair of double-strand breaks (DSBs) on the eimerian chromosomes as they synapse and desynapse. Structural Maintenance of Chromosome protein SMC3, which plays a role in synaptonemal complex formation, was labeled to identify initiation and progress of chromosome synapsis and desynapsis in parallel with the appearance and disappearance of RAD51 foci. Antibodies directed against RAD51 and cohesin subunit SMC3 proteins were labeled with either fluorescence or colloidal gold to visualize RAD51 protein foci and synaptonemal complexes. RAD51 protein localization during prophase I was studied on meiotic chromosomes spreads obtained from oocysts at different points in time after the start of sporulation. The present findings showed that foci detected with the antibody directed against RAD51 protein first appeared at the pre-leptotene stage before homologous chromosomes began pairing. Subsequently, the foci were detected in association with the lateral elements at the precise sites where synapsis were in progress. These findings lead us to suggest that in E. tenella, homologous chromosome pairing was a DSB-dependent mechanism and reinforced the participation of RAD51 protein in meiotic homology search, alignment and pairing of chromosomes. Copyright © 2010 Elsevier B.V. All rights reserved.

  18. Secondary Somatic Mutations Restoring RAD51C and RAD51D Associated with Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma

    PubMed Central

    Kondrashova, Olga; Nguyen, Minh; Shield-Artin, Kristy; Tinker, Anna V.; Teng, Nelson N.H.; Harrell, Maria I.; Kuiper, Michael J.; Ho, Gwo-Yaw; Barker, Holly; Jasin, Maria; Prakash, Rohit; Kass, Elizabeth M.; Sullivan, Meghan R.; Brunette, Gregory J.; Bernstein, Kara A.; Coleman, Robert L.; Floquet, Anne; Friedlander, Michael; Kichenadasse, Ganessan; O'Malley, David M.; Oza, Amit; Sun, James; Robillard, Liliane; Maloney, Lara; Giordano, Heidi; Wakefield, Matthew J.; Kaufmann, Scott H.; Simmons, Andrew D.; Harding, Thomas C.; Raponi, Mitch; McNeish, Iain A.; Swisher, Elizabeth M.; Lin, Kevin K.; Scott, Clare L.

    2017-01-01

    High-grade epithelial ovarian carcinomas containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in BRCA1, RAD51C, or RAD51D was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51C. In vitro complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations. Significance Analyses of primary and secondary mutations in RAD51C and RAD51D provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies. PMID:28588062

  19. Recovery of deficient homologous recombination in Brca2-depleted mouse cells by wild-type Rad51 expression.

    PubMed

    Lee, Shauna A; Roques, Céline; Magwood, Alissa C; Masson, Jean-Yves; Baker, Mark D

    2009-02-01

    The BRCA2 tumor suppressor is important in maintaining genomic stability. BRCA2 is proposed to control the availability, cellular localization and DNA binding activity of the central homologous recombination protein, RAD51, with loss of BRCA2 resulting in defective homologous recombination. Nevertheless, the roles of BRCA2 in regulating RAD51 and how other proteins implicated in RAD51 regulation, such as RAD52 and RAD54 function relative to BRCA2 is not known. In this study, we tested whether defective homologous recombination in Brca2-depleted mouse hybridoma cells could be rectified by expression of mouse Rad51 or the Rad51-interacting mouse proteins, Rad52 and Rad54. In the Brca2-depleted cells, defective homologous recombination can be restored by over-expression of wild-type mouse Rad51, but not mouse Rad52 or Rad54. Correction of the homologous recombination defect requires Rad51 ATPase activity. A sizeable fraction ( approximately 50%) of over-expressed wild-type Rad51 is nuclear localized. The restoration of homologous recombination in the presence of a low (i.e., non-functional) level of Brca2 by wild-type Rad51 over-expression is unexpected. We suggest that Rad51 may access the nuclear compartment in a Brca2-independent manner and when Rad51 is over-expressed, the normal requirement for Brca2 control over Rad51 function in homologous recombination is dispensable. Our studies support loss of Rad51 function as a critical underlying factor in the homologous recombination defect in the Brca2-depleted cells.

  20. Suppression of OsRAD51D results in defects in reproductive development in rice (Oryza sativa L.).

    PubMed

    Byun, Mi Young; Kim, Woo Taek

    2014-07-01

    The cellular roles of RAD51 paralogs in somatic and reproductive growth have been extensively described in a wide range of animal systems and, to a lesser extent, in Arabidopsis, a dicot model plant. Here, the OsRAD51D gene was identified and characterized in rice (Oryza sativa L.), a monocot model crop. In the rice genome, three alternative OsRAD51D mRNA splicing variants, OsRAD51D.1, OsRAD51D.2, and OsRAD51D.3, were predicted. Yeast two-hybrid studies, however, showed that only OsRAD51D.1 interacted with OsRAD51B and OsRAD51C paralogs, suggesting that OsRAD51D.1 is a functional OsRAD51D protein in rice. Loss-of-function osrad51d mutant rice plants displayed normal vegetative growth. However, the mutant plants were defective in reproductive growth, resulting in sterile flowers. Homozygous osrad51d mutant flowers exhibited impaired development of lemma and palea and contained unusual numbers of stamens and stigmas. During early meiosis, osrad51d pollen mother cells (PMCs) failed to form normal homologous chromosome pairings. In subsequent meiotic progression, mutant PMCs represented fragmented chromosomes. The osrad51d pollen cells contained numerous abnormal micro-nuclei that resulted in malfunctioning pollen. The abnormalities of heterozygous mutant and T2 Ubi:RNAi-OsRAD51D RNAi-knock-down transgenic plants were intermediate between those of wild type and homozygous mutant plants. The osrad51d and Ubi:RNAi-OsRAD51D plants contained longer telomeres compared with wild type plants, indicating that OsRAD51D is a negative factor for telomere lengthening. Overall, these results suggest that OsRAD51D plays a critical role in reproductive growth in rice. This essential function of OsRAD51D is distinct from Arabidopsis, in which AtRAD51D is not an essential factor for meiosis or reproductive development. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  1. Disparate requirements for the Walker A and B ATPase motifs of human RAD51D in homologous recombination.

    PubMed

    Wiese, Claudia; Hinz, John M; Tebbs, Robert S; Nham, Peter B; Urbin, Salustra S; Collins, David W; Thompson, Larry H; Schild, David

    2006-01-01

    In vertebrates, homologous recombinational repair (HRR) requires RAD51 and five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C and RAD51D) that all contain conserved Walker A and B ATPase motifs. In human RAD51D we examined the requirement for these motifs in interactions with XRCC2 and RAD51C, and for survival of cells in response to DNA interstrand crosslinks (ICLs). Ectopic expression of wild-type human RAD51D or mutants having a non-functional A or B motif was used to test for complementation of a rad51d knockout hamster CHO cell line. Although A-motif mutants complement very efficiently, B-motif mutants do not. Consistent with these results, experiments using the yeast two- and three-hybrid systems show that the interactions between RAD51D and its XRCC2 and RAD51C partners also require a functional RAD51D B motif, but not motif A. Similarly, hamster Xrcc2 is unable to bind to the non-complementing human RAD51D B-motif mutants in co-immunoprecipitation assays. We conclude that a functional Walker B motif, but not A motif, is necessary for RAD51D's interactions with other paralogs and for efficient HRR. We present a model in which ATPase sites are formed in a bipartite manner between RAD51D and other RAD51 paralogs.

  2. Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

    PubMed

    Callender, Tracy L; Laureau, Raphaelle; Wan, Lihong; Chen, Xiangyu; Sandhu, Rima; Laljee, Saif; Zhou, Sai; Suhandynata, Ray T; Prugar, Evelyn; Gaines, William A; Kwon, YoungHo; Börner, G Valentin; Nicolas, Alain; Neiman, Aaron M; Hollingsworth, Nancy M

    2016-08-01

    During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1.

  3. Rad51 Interacts with Non-structural 3 Protein of Hepatitis C Virus and Regulates Viral Production

    PubMed Central

    Son, Kidong; Nguyen, Tram T. T.; Choi, Jae-Woong; Pham, Long V.; Luong, Trang T. D.; Lim, Yun-Sook; Hwang, Soon B.

    2017-01-01

    Hepatitis C virus (HCV) is a leading cause of chronic liver disease affecting over 170 million people worldwide. Chronic infection with HCV progresses to liver fibrosis, cirrhosis, and hepatocellular carcinoma. HCV exploits host cellular factors for viral propagation. To investigate the cellular factors required for HCV propagation, we screened a siRNA library targeting human cell cycle genes using cell culture grown HCV-infected cells. In the present study, we selected and characterized a gene encoding Rad51. Rad51, a member of a conserved recombinase family, is an essential factor for homologous recombination and repair of double-strand DNA breaks. We demonstrated that siRNA-mediated knockdown of Rad51 significantly inhibited HCV propagation without affecting HCV RNA replication. Silencing of Rad51 impaired secretion of infectious HCV particles and thus intracellular viruses were accumulated. We showed that HCV NS3 specifically interacted with Rad51 and accumulated Rad51 in the cytosol. Furthermore, Rad51 was coprecipitated with NS3 and HCV RNA. By employing membrane flotation and protease protection assays, we also demonstrated that Rad51 was co-fractionated with HCV NS3 on the lipid raft. These data indicate that Rad51 may be a component of the HCV RNA replication complex. Collectively, these data suggest that HCV may exploit cellular Rad51 to promote viral propagation and thus Rad51 may be a potential therapeutic target for HCV. PMID:28729862

  4. Dynamic changes in Rad51 distribution on chromatin during meiosis in male and female vertebrates.

    PubMed

    Ashley, T; Plug, A W; Xu, J; Solari, A J; Reddy, G; Golub, E I; Ward, D C

    1995-10-01

    Antibodies against human Rad51 protein were used to examine the distribution of Rad51 on meiotic chromatin in mouse spermatocytes and oocytes as well as chicken oocytes during sequential stages of meiosis. We observed the following dynamic changes in distribution of Rad51 during meiosis: (1) in early leptotene nuclei there are multiple, apparently randomly distributed, foci that by late leptonema become organized into tracks of foci. (2) These foci persist into zygonema, but most foci are now localized on Rad51-positive axes that correspond to lateral elements of the synaptonemal complex. As homologs synapse foci from homologous axes fuse. The distribution and involvement of Rad51 foci as contact points between homologs suggest that they may be components to early recombination nodules. (3) As pachynema progresses the number of foci drops dramatically; the temporal occurrence (mice) and physical and numerical distribution of foci on axes (chickens) suggest that they may be a component of late recombination nodules. (4) In early pachynema there are numerous Rad51 foci on the single axis of the X (mouse spermatocytes) or the Z (chicken oocytes) chromosomes that neither pair, nor recombine. (5) In late pachynema in mouse spermatocytes, but not oocytes, the Rad51 signal is preferentially enhanced at both ends of all the bivalents. As bivalents in spermatocytes, but not oocytes, begin to desynapse at diplonema they are often held together at these Rad51-positive termini. These observations parallel observations that recombination rates are exceptionally high near chromosome ends in male but not female eutherian mammals. (6) From diakinesis through metaphase I, Rad51 protein is detected as low-intensity fluorescent doublets that localize with CREST-specific antigens (kinetochores), suggesting that Rad51 participates, at least as a structural component of the materials involved, in sister kinetochore cohesiveness. Finally, the changes in Rad51 distribution during meiosis

  5. Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly.

    PubMed

    Tojkander, Sari; Gateva, Gergana; Husain, Amjad; Krishnan, Ramaswamy; Lappalainen, Pekka

    2015-12-10

    Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells.

  6. ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage

    PubMed Central

    McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

    2005-01-01

    Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions. PMID:15933716

  7. ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage.

    PubMed

    McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

    2005-07-06

    Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions.

  8. Accessory factors promote AlfA-dependent plasmid segregation by regulating filament nucleation, disassembly, and bundling

    PubMed Central

    Polka, Jessica K.; Kollman, Justin M.; Mullins, R. Dyche

    2014-01-01

    In bacteria, some plasmids are partitioned to daughter cells by assembly of actin-like proteins (ALPs). The best understood ALP, ParM, has a core set of biochemical properties that contributes to its function, including dynamic instability, spontaneous nucleation, and bidirectional elongation. AlfA, an ALP that pushes plasmids apart in Bacillus, relies on a different set of underlying properties to segregate DNA. AlfA elongates unidirectionally and is not dynamically unstable; its assembly and disassembly are regulated by a cofactor, AlfB. Free AlfB breaks up AlfA bundles and promotes filament turnover. However, when AlfB is bound to the centromeric DNA sequence, parN, it forms a segrosome complex that nucleates and stabilizes AlfA filaments. When reconstituted in vitro, this system creates polarized, motile comet tails that associate by antiparallel filament bundling to form bipolar, DNA-segregating spindles. PMID:24481252

  9. RAD51 and RTEL1 compensate telomere loss in the absence of telomerase

    PubMed Central

    Olivier, Margaux; Charbonnel, Cyril; Amiard, Simon

    2018-01-01

    Abstract Replicative erosion of telomeres is naturally compensated by telomerase and studies in yeast and vertebrates show that homologous recombination can compensate for the absence of telomerase. We show that RAD51 protein, which catalyzes the key strand-invasion step of homologous recombination, is localized at Arabidopsis telomeres in absence of telomerase. Blocking the strand-transfer activity of the RAD51 in telomerase mutant plants results in a strikingly earlier onset of developmental defects, accompanied by increased numbers of end-to-end chromosome fusions. Imposing replication stress through knockout of RNaseH2 increases numbers of chromosome fusions and reduces the survival of these plants deficient for telomerase and homologous recombination. This finding suggests that RAD51-dependent homologous recombination acts as an essential backup to the telomerase for compensation of replicative telomere loss to ensure genome stability. Furthermore, we show that this positive role of RAD51 in telomere stability is dependent on the RTEL1 helicase. We propose that a RAD51 dependent break-induced replication process is activated in cells lacking telomerase activity, with RTEL1 responsible for D-loop dissolution after telomere replication. PMID:29346668

  10. RAD51 and RTEL1 compensate telomere loss in the absence of telomerase.

    PubMed

    Olivier, Margaux; Charbonnel, Cyril; Amiard, Simon; White, Charles I; Gallego, Maria E

    2018-03-16

    Replicative erosion of telomeres is naturally compensated by telomerase and studies in yeast and vertebrates show that homologous recombination can compensate for the absence of telomerase. We show that RAD51 protein, which catalyzes the key strand-invasion step of homologous recombination, is localized at Arabidopsis telomeres in absence of telomerase. Blocking the strand-transfer activity of the RAD51 in telomerase mutant plants results in a strikingly earlier onset of developmental defects, accompanied by increased numbers of end-to-end chromosome fusions. Imposing replication stress through knockout of RNaseH2 increases numbers of chromosome fusions and reduces the survival of these plants deficient for telomerase and homologous recombination. This finding suggests that RAD51-dependent homologous recombination acts as an essential backup to the telomerase for compensation of replicative telomere loss to ensure genome stability. Furthermore, we show that this positive role of RAD51 in telomere stability is dependent on the RTEL1 helicase. We propose that a RAD51 dependent break-induced replication process is activated in cells lacking telomerase activity, with RTEL1 responsible for D-loop dissolution after telomere replication.

  11. Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly

    PubMed Central

    Tojkander, Sari; Gateva, Gergana; Husain, Amjad; Krishnan, Ramaswamy; Lappalainen, Pekka

    2015-01-01

    Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells. DOI: http://dx.doi.org/10.7554/eLife.06126.001 PMID:26652273

  12. The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair.

    PubMed

    Cukras, Scott; Lee, Euiho; Palumbo, Emily; Benavidez, Pamela; Moldovan, George-Lucian; Kee, Younghoon

    2016-10-01

    USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.

  13. The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair

    PubMed Central

    Cukras, Scott; Lee, Euiho; Palumbo, Emily; Benavidez, Pamela; Moldovan, George-Lucian; Kee, Younghoon

    2016-01-01

    ABSTRACT USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1. PMID:27463890

  14. NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection

    PubMed Central

    Abeyta, Antonio; Castella, Maria; Jacquemont, Celine; Taniguchi, Toshiyasu

    2017-01-01

    ABSTRACT Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51. PMID:27892797

  15. NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection.

    PubMed

    Abeyta, Antonio; Castella, Maria; Jacquemont, Celine; Taniguchi, Toshiyasu

    2017-02-16

    Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51.

  16. Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation

    PubMed Central

    Zelensky, Alex N.; Sanchez, Humberto; Ristic, Dejan; Vidic, Iztok; van Rossum-Fikkert, Sari E.; Essers, Jeroen; Wyman, Claire; Kanaar, Roland

    2013-01-01

    Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Biochemical and structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair. PMID:23666627

  17. Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale

    PubMed Central

    Yu, Xiong; Egelman, Edward H.

    2010-01-01

    Proteins in the RecA/Rad51/RadA family form nucleoprotein filaments on DNA that catalyze a strand exchange reaction as part of homologous genetic recombination. Because of the centrality of this system to many aspects of DNA repair, the generation of genetic diversity, and cancer when this system fails or is not properly regulated, these filaments have been the object of many biochemical and biophysical studies. A recent paper has argued that the human Dmc1 protein, a meiotic homolog of bacterial RecA and human Rad51, forms filaments on single stranded DNA with ∼ 9 subunits per turn in contrast to the filaments formed on double stranded DNA with ∼ 6.4 subunits per turn, and that the stoichiometry of DNA binding is different between these two filaments. We show using scanning transmission electron microscopy (STEM) that the Dmc1 filament formed on single stranded DNA has a mass per unit length expected from ∼ 6.5 subunits per turn. More generally, we show how ambiguities in helical symmetry determination can generate incorrect solutions, and why one sometimes must use other techniques, such as biochemistry, metal shadowing, or STEM to resolve these ambiguities. While three-dimensional reconstruction of helical filaments from EM images is a powerful tool, the intrinsic ambiguities that may be present with limited resolution are not sufficiently appreciated. PMID:20600108

  18. Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

    PubMed Central

    Konomura, Naoto; Arai, Naoto; Shinohara, Takeshi; Kobayashi, Jun; Iwasaki, Wakana; Ikawa, Shukuko; Kusano, Kohji; Shibata, Takehiko

    2017-01-01

    RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2–5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo. PMID:27794044

  19. Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale.

    PubMed

    Yu, Xiong; Egelman, Edward H

    2010-08-20

    Proteins in the RecA/Rad51/RadA family form nucleoprotein filaments on DNA that catalyze a strand exchange reaction as part of homologous genetic recombination. Because of the centrality of this system to many aspects of DNA repair, the generation of genetic diversity, and cancer when this system fails or is not properly regulated, these filaments have been the object of many biochemical and biophysical studies. A recent paper has argued that the human Dmc1 protein, a meiotic homolog of bacterial RecA and human Rad51, forms filaments on single-stranded DNA with approximately 9 subunits per turn in contrast to the filaments formed on double-stranded DNA with approximately 6.4 subunits per turn and that the stoichiometry of DNA binding is different between these two filaments. We show using scanning transmission electron microscopy that the Dmc1 filament formed on single-stranded DNA has a mass per unit length expected from approximately 6.5 subunits per turn. More generally, we show how ambiguities in helical symmetry determination can generate incorrect solutions and why one sometimes must use other techniques, such as biochemistry, metal shadowing, or scanning transmission electron microscopy, to resolve these ambiguities. While three-dimensional reconstruction of helical filaments from EM images is a powerful tool, the intrinsic ambiguities that may be present with limited resolution are not sufficiently appreciated. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  20. Engineering Archeal Surrogate Systems for the Development of Protein-Protein Interaction Inhibitors against Human RAD51.

    PubMed

    Moschetti, Tommaso; Sharpe, Timothy; Fischer, Gerhard; Marsh, May E; Ng, Hong Kin; Morgan, Matthew; Scott, Duncan E; Blundell, Tom L; R Venkitaraman, Ashok; Skidmore, John; Abell, Chris; Hyvönen, Marko

    2016-11-20

    Protein-protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to "humanise" thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from Pyrococcus furiosus. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Differing Requirements for RAD51 and DMC1 in Meiotic Pairing of Centromeres and Chromosome Arms in Arabidopsis thaliana

    PubMed Central

    Da Ines, Olivier; Abe, Kiyomi; Goubely, Chantal; Gallego, Maria Eugenia; White, Charles I.

    2012-01-01

    During meiosis homologous chromosomes pair, recombine, and synapse, thus ensuring accurate chromosome segregation and the halving of ploidy necessary for gametogenesis. The processes permitting a chromosome to pair only with its homologue are not fully understood, but successful pairing of homologous chromosomes is tightly linked to recombination. In Arabidopsis thaliana, meiotic prophase of rad51, xrcc3, and rad51C mutants appears normal up to the zygotene/pachytene stage, after which the genome fragments, leading to sterility. To better understand the relationship between recombination and chromosome pairing, we have analysed meiotic chromosome pairing in these and in dmc1 mutant lines. Our data show a differing requirement for these proteins in pairing of centromeric regions and chromosome arms. No homologous pairing of mid-arm or distal regions was observed in rad51, xrcc3, and rad51C mutants. However, homologous centromeres do pair in these mutants and we show that this does depend upon recombination, principally on DMC1. This centromere pairing extends well beyond the heterochromatic centromere region and, surprisingly, does not require XRCC3 and RAD51C. In addition to clarifying and bringing the roles of centromeres in meiotic synapsis to the fore, this analysis thus separates the roles in meiotic synapsis of DMC1 and RAD51 and the meiotic RAD51 paralogs, XRCC3 and RAD51C, with respect to different chromosome domains. PMID:22532804

  2. Dmc1 Functions in a Saccharomyces Cerevisiae Meiotic Pathway That Is Largely Independent of the Rad51 Pathway

    PubMed Central

    Dresser, M. E.; Ewing, D. J.; Conrad, M. N.; Dominguez, A. M.; Barstead, R.; Jiang, H.; Kodadek, T.

    1997-01-01

    Meiotic recombination in the yeast Saccharomyces cerevisiae requires two similar recA-like proteins, Dmc1p and Rad51p. A screen for dominant meiotic mutants provided DMC1-G126D, a dominant allele mutated in the conserved ATP-binding site (specifically, the A-loop motif) that confers a null phenotype. A recessive null allele, dmc1-K69E, was isolated as an intragenic suppressor of DMC1-G126D. Dmc1-K69Ep, unlike Dmc1p, does not interact homotypically in a two-hybrid assay, although it does interact with other fusion proteins identified by two-hybrid screen with Dmc1p. Dmc1p, unlike Rad51p, does not interact in the two-hybrid assay with Rad52p or Rad54p. However, Dmc1p does interact with Tid1p, a Rad54p homologue, with Tid4p, a Rad16p homologue, and with other fusion proteins that do not interact with Rad51p, suggesting that Dmc1p and Rad51p function in separate, though possibly overlapping, recombinational repair complexes. Epistasis analysis suggests that DMC1 and RAD51 function in separate pathways responsible for meiotic recombination. Taken together, our results are consistent with a requirement for DMC1 for meiosis-specific entry of DNA double-strand break ends into chromatin. Interestingly, the pattern on CHEF gels of chromosome fragments that result from meiotic DNA double-strand break formation is different in DMC1 mutant strains from that seen in rad50S strains. PMID:9335591

  3. Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31.

    PubMed

    Chappell, William H; Gautam, Dipendra; Ok, Suzan T; Johnson, Bryan A; Anacker, Daniel C; Moody, Cary A

    2015-12-23

    High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral

  4. Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

    PubMed Central

    Chappell, William H.; Gautam, Dipendra; Ok, Suzan T.; Johnson, Bryan A.; Anacker, Daniel C.

    2015-01-01

    ABSTRACT High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. IMPORTANCE Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for

  5. 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

  6. Inhibition of RAD51 by siRNA and Resveratrol Sensitizes Cancer Stem Cells Derived from HeLa Cell Cultures to Apoptosis

    PubMed Central

    Ruíz, Graciela; Valencia-González, Heriberto A.; León-Galicia, Ismael; García-Villa, Enrique

    2018-01-01

    Cervical cancer is the second most frequent tumor type in women worldwide with cases developing clinical recurrence, metastasis, and chemoresistance. The cancer stem cells (CSC) may be implicated in tumor resistance to therapy. RESveratrol (RES), a natural compound, is an antioxidant with multiple beneficial activities. We previously determined that the expression of RAD51 is decreased by RES. The aim of our study was to examine molecular mechanism by which CSC from HeLa cultures exhibit chemoresistance. We hypothesized CSC repair more efficiently DNA breaks and that RAD51 plays an important role in this mechanism. We found that CSC, derived from cervical cancer cell lines, overexpress RAD51 and are less sensitive to Etoposide (VP16). We inhibited RAD51 in CSC-enriched cultures using RES or siRNA against RAD51 messenger RNA and observed a decrease in cell viability and induction of apoptosis when treated simultaneously with VP16. In addition, we found that inhibition of RAD51 expression using RES also sensitizes CSC to VP16 treatment. Our results suggest that resveratrol is effective to sensitize cervical CSC because of RAD51 inhibition, targeting high RAD51 expressing CD49f-positive cells, which supports the possible therapeutic application of RES as a novel agent to treat cancer. PMID:29681946

  7. Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs

    PubMed Central

    Lopes, Anne; Amarir-Bouhram, Jihane; Faure, Guilhem; Petit, Marie-Agnès; Guerois, Raphaël

    2010-01-01

    Homologous recombination is a key in contributing to bacteriophages genome repair, circularization and replication. No less than six kinds of recombinase genes have been reported so far in bacteriophage genomes, two (UvsX and Gp2.5) from virulent, and four (Sak, Redβ, Erf and Sak4) from temperate phages. Using profile–profile comparisons, structure-based modelling and gene-context analyses, we provide new views on the global landscape of recombinases in 465 bacteriophages. We show that Sak, Redβ and Erf belong to a common large superfamily adopting a shortcut Rad52-like fold. Remote homologs of Sak4 are predicted to adopt a shortcut Rad51/RecA fold and are discovered widespread among phage genomes. Unexpectedly, within temperate phages, gene-context analyses also pinpointed the presence of distant Gp2.5 homologs, believed to be restricted to virulent phages. All in all, three major superfamilies of phage recombinases emerged either related to Rad52-like, Rad51-like or Gp2.5-like proteins. For two newly detected recombinases belonging to the Sak4 and Gp2.5 families, we provide experimental evidence of their recombination activity in vivo. Temperate versus virulent lifestyle together with the importance of genome mosaicism is discussed in the light of these novel recombinases. Screening for these recombinases in genomes can be performed at http://biodev.extra.cea.fr/virfam. PMID:20194117

  8. Breast Imaging-Reporting and Data System (BI-RADS) classification in 51 excised palpable pediatric breast masses.

    PubMed

    Koning, Jeffrey L; Davenport, Katherine P; Poole, Patricia S; Kruk, Peter G; Grabowski, Julia E

    2015-10-01

    The American College of Radiology Breast Imaging Reporting and Data System (BI-RADS) classification was developed to risk stratify breast lesions and guide surgical management based on imaging. Previous studies validating BI-RADS for US do not include pediatric patients. Most pediatric breast masses present as palpable lesions and frequently undergo ultrasound, which is often accompanied with a BI-RADS classification. Our study aimed to correlate BI-RADS with pathology findings to assess applicability of the classification system to pediatric patients. We performed a retrospective review of all patients who underwent excision of a breast mass at a single center from July 2010 to November 2013. We identified all patients who underwent preoperative ultrasound with BI-RADS classification. Demographic data, imaging results, and surgical pathology were analyzed and correlated. A total of 119 palpable masses were excised from 105 pediatric patients during the study period. Of 119 masses, 81 had preoperative ultrasound, and BI-RADS categories were given to 51 masses. Of these 51, all patients were female and the average age was 15.9 years. BI-RADS 4 was given to 25 of 51 masses (49%), and 100% of these lesions had benign pathology, the most common being fibroadenoma. Treatment algorithm based on BI-RADS classification may not be valid in pediatric patients. In this study, all patients with a BI-RADS 4 lesion had benign pathology. BI-RADS classification may overstate the risk of malignancy or need for biopsy in this population. Further validation of BI-RADS classification with large scale studies is needed in pediatric and adolescent patients. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Loss of the homologous recombination gene rad51 leads to Fanconi anemia-like symptoms in zebrafish.

    PubMed

    Botthof, Jan Gregor; Bielczyk-Maczyńska, Ewa; Ferreira, Lauren; Cvejic, Ana

    2017-05-30

    RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant-negative mutations. FA is a hereditary DNA-repair disorder characterized by various congenital abnormalities, progressive bone marrow failure, and cancer predisposition. In this report, we describe a viable vertebrate model of RAD51 loss. Zebrafish rad51 loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to cross-linking agents, and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Comutation of p53 was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

  10. Human CST Facilitates Genome-wide RAD51 Recruitment to GC-Rich Repetitive Sequences in Response to Replication Stress.

    PubMed

    Chastain, Megan; Zhou, Qing; Shiva, Olga; Fadri-Moskwik, Maria; Whitmore, Leanne; Jia, Pingping; Dai, Xueyu; Huang, Chenhui; Ye, Ping; Chai, Weihang

    2016-08-02

    The telomeric CTC1/STN1/TEN1 (CST) complex has been implicated in promoting replication recovery under replication stress at genomic regions, yet its precise role is unclear. Here, we report that STN1 is enriched at GC-rich repetitive sequences genome-wide in response to hydroxyurea (HU)-induced replication stress. STN1 deficiency exacerbates the fragility of these sequences under replication stress, resulting in chromosome fragmentation. We find that upon fork stalling, CST proteins form distinct nuclear foci that colocalize with RAD51. Furthermore, replication stress induces physical association of CST with RAD51 in an ATR-dependent manner. Strikingly, CST deficiency diminishes HU-induced RAD51 foci formation and reduces RAD51 recruitment to telomeres and non-telomeric GC-rich fragile sequences. Collectively, our findings establish that CST promotes RAD51 recruitment to GC-rich repetitive sequences in response to replication stress to facilitate replication restart, thereby providing insights into the mechanism underlying genome stability maintenance. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  11. RPA and Rad51 constitute a cell intrinsic mechanism to protect the cytosol from self DNA.

    PubMed

    Wolf, Christine; Rapp, Alexander; Berndt, Nicole; Staroske, Wolfgang; Schuster, Max; Dobrick-Mattheuer, Manuela; Kretschmer, Stefanie; König, Nadja; Kurth, Thomas; Wieczorek, Dagmar; Kast, Karin; Cardoso, M Cristina; Günther, Claudia; Lee-Kirsch, Min Ae

    2016-05-27

    Immune recognition of cytosolic DNA represents a central antiviral defence mechanism. Within the host, short single-stranded DNA (ssDNA) continuously arises during the repair of DNA damage induced by endogenous and environmental genotoxic stress. Here we show that short ssDNA traverses the nuclear membrane, but is drawn into the nucleus by binding to the DNA replication and repair factors RPA and Rad51. Knockdown of RPA and Rad51 enhances cytosolic leakage of ssDNA resulting in cGAS-dependent type I IFN activation. Mutations in the exonuclease TREX1 cause type I IFN-dependent autoinflammation and autoimmunity. We demonstrate that TREX1 is anchored within the outer nuclear membrane to ensure immediate degradation of ssDNA leaking into the cytosol. In TREX1-deficient fibroblasts, accumulating ssDNA causes exhaustion of RPA and Rad51 resulting in replication stress and activation of p53 and type I IFN. Thus, the ssDNA-binding capacity of RPA and Rad51 constitutes a cell intrinsic mechanism to protect the cytosol from self DNA.

  12. Nanoscopic exclusion between Rad51 and 53BP1 after ion irradiation in human HeLa cells

    NASA Astrophysics Data System (ADS)

    Reindl, Judith; Drexler, Guido A.; Girst, Stefanie; Greubel, Christoph; Siebenwirth, Christian; Drexler, Sophie E.; Dollinger, Günther; Friedl, Anna A.

    2015-12-01

    Many proteins involved in detection, signalling and repair of DNA double-strand breaks (DSB) accumulate in large number in the vicinity of DSB sites, forming so called foci. Emerging evidence suggests that these foci are sub-divided in structural or functional domains. We use stimulated emission depletion (STED) microscopy to investigate localization of mediator protein 53BP1 and recombination factor Rad51 after irradiation of cells with low linear energy transfer (LET) protons or high LET carbon ions. With a resolution better than 100 nm, STED microscopy and image analysis using a newly developed analyzing algorithm, the reduced product of the differences from the mean, allowed us to demonstrate that with both irradiation types Rad51 occupies spherical regions of about 200 nm diameter. These foci locate within larger 53BP1 accumulations in regions of local 53BP1 depletion, similar to what has been described for the localization of Brca1, CtIP and RPA. Furthermore, localization relative to 53BP1 and size of Rad51 foci was not different after irradiation with low and high LET radiation. As expected, 53BP1 foci induced by low LET irradiation mostly contained one Rad51 focal structure, while after high LET irradiation, most foci contained >1 Rad51 accumulation.

  13. Homologous and Homeologous Intermolecular Gene Conversion Are Not Differentially Affected by Mutations in the DNA Damage or the Mismatch Repair Genes Rad1, Rad50, Rad51, Rad52, Rad54, Pms1 and Msh2

    PubMed Central

    Porter, G.; Westmoreland, J.; Priebe, S.; Resnick, M. A.

    1996-01-01

    Mismatch repair (MMR) genes or genes involved in both DNA damage repair and homologous recombination might affect homeologous vs. homologous recombination differentially. Spontaneous mitotic gene conversion between a chromosome and a homologous or homeologous donor sequence (14% diverged) on a single copy plasmid was examined in wild-type Saccharomyces cerevisiae strains and in MMR or DNA damage repair mutants. Homologous recombination in rad51, rad52 and rad54 mutants was considerably reduced, while there was little effect of rad1, rad50, pms1 and msh2 null mutations. DNA divergence resulted in no differential effect on recombination rates in the wild type or the mutants; there was only a five- to 10-fold reduction in homeologous relative to homologous recombination regardless of background. Since DNA divergence is known to affect recombination in some systems, we propose that differences in the role of MMR depends on the mode of recombination and/or the level of divergence. Based on analysis of the recombination breakpoints, there is a minimum of three homologous bases required at a recombination junction. A comparison of Rad(+) vs. rad52 strains revealed that while all conversion tracts are continuous, elimination of RAD52 leads to the appearance of a novel class of very short conversion tracts. PMID:8725224

  14. NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability

    DOE PAGES

    Parplys, Ann C.; Zhao, Weixing; Sharma, Neelam; ...

    2015-08-31

    NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintainingmore » wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Finally, our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.« less

  15. A novel Fanconi anaemia subtype associated with a dominant-negative mutation in RAD51

    PubMed Central

    Ameziane, Najim; May, Patrick; Haitjema, Anneke; van de Vrugt, Henri J.; van Rossum-Fikkert, Sari E.; Ristic, Dejan; Williams, Gareth J.; Balk, Jesper; Rockx, Davy; Li, Hong; Rooimans, Martin A.; Oostra, Anneke B.; Velleuer, Eunike; Dietrich, Ralf; Bleijerveld, Onno B.; Maarten Altelaar, A. F.; Meijers-Heijboer, Hanne; Joenje, Hans; Glusman, Gustavo; Roach, Jared; Hood, Leroy; Galas, David; Wyman, Claire; Balling, Rudi; den Dunnen, Johan; de Winter, Johan P.; Kanaar, Roland; Gelinas, Richard; Dorsman, Josephine C.

    2015-01-01

    Fanconi anaemia (FA) is a hereditary disease featuring hypersensitivity to DNA cross-linker-induced chromosomal instability in association with developmental abnormalities, bone marrow failure and a strong predisposition to cancer. A total of 17 FA disease genes have been reported, all of which act in a recessive mode of inheritance. Here we report on a de novo g.41022153G>A; p.Ala293Thr (NM_002875) missense mutation in one allele of the homologous recombination DNA repair gene RAD51 in an FA-like patient. This heterozygous mutation causes a novel FA subtype, ‘FA-R', which appears to be the first subtype of FA caused by a dominant-negative mutation. The patient, who features microcephaly and mental retardation, has reached adulthood without the typical bone marrow failure and paediatric cancers. Together with the recent reports on RAD51-associated congenital mirror movement disorders, our results point to an important role for RAD51-mediated homologous recombination in neurodevelopment, in addition to DNA repair and cancer susceptibility. PMID:26681308

  16. RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.

    PubMed

    Inano, Shojiro; Sato, Koichi; Katsuki, Yoko; Kobayashi, Wataru; Tanaka, Hiroki; Nakajima, Kazuhiro; Nakada, Shinichiro; Miyoshi, Hiroyuki; Knies, Kerstin; Takaori-Kondo, Akifumi; Schindler, Detlev; Ishiai, Masamichi; Kurumizaka, Hitoshi; Takata, Minoru

    2017-06-01

    RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. RAD51 potentiates synergistic effects of chemotherapy with PCI-24781 and cis-diamminedichloroplatinum on gastric cancer

    PubMed Central

    He, Wei-Ling; Li, Yu-Huang; Hou, Wei-Jian; Ke, Zun-Fu; Chen, Xin-Lin; Lu, Li-Ya; Cai, Shi-Rong; Song, Wu; Zhang, Chang-Hua; He, Yu-Long

    2014-01-01

    AIM: To explore the efficacy of PCI-24781, a broad-spectrum, hydroxamic acid-derived histone deacetylase inhibitor, in the treatment of gastric cancer (GC). METHODS: With or without treatment of PCI-24781 and/or cis-diamminedichloroplatinum (CDDP), GC cell lines were subjected to functional analysis, including cell growth, apoptosis and clonogenic assays. Chromatin immunoprecipitation and luciferase reporter assays were used to determine the interacting molecules and the activity of the enzyme. An in vivo study was carried out in GC xenograft mice. Cell culture-based assays were represented as mean ± SD. ANOVA tests were used to assess differences across groups. All pairwise comparisons between tumor weights among treatment groups were made using the Tukey-Kramer method for multiple comparison adjustment to control experimental-wise type I error rates. Significance was set at P < 0.05. RESULTS: PCI-24781 significantly reduced the growth of the GC cells, enhanced cell apoptosis and suppressed clonogenicity, and these effects synergized with the effects of CDDP. PCI-24781 modulated the cell cycle and significantly reduced the expression of RAD51, which is related to homologous recombination. Depletion of RAD51 augmented the biological functions of PCI-24781, CDDP and the combination treatment, whereas overexpressing RAD51 had the opposite effects. Increased binding of the transcription suppressor E2F4 on the RAD51 promoter appeared to play a major role in these processes. Furthermore, significant suppression of tumor growth and weight in vivo was obtained following PCI-24781 treatment, which synergized with the anticancer effect of CDDP. CONCLUSION: These data suggest that RAD51 potentiates the synergistic effects of chemotherapy with PCI-24781 and CDDP on GC. PMID:25110436

  18. Cofilin-2 controls actin filament length in muscle sarcomeres

    PubMed Central

    Kremneva, Elena; Makkonen, Maarit H.; Skwarek-Maruszewska, Aneta; Gateva, Gergana; Michelot, Alphee; Dominguez, Roberto; Lappalainen, Pekka

    2014-01-01

    SUMMARY ADF/cofilins drive cytoskeletal dynamics by promoting the disassembly of ‘aged’ ADP-actin filaments. Mammals express several ADF/cofilin isoforms, but their specific biochemical activities and cellular functions have not been studied in detail. Here we demonstrate that the muscle-specific isoform cofilin-2 promotes actin filament disassembly in sarcomeres to control the precise length of thin filaments in the contractile apparatus. In contrast to other isoforms, cofilin-2 efficiently binds and disassembles both ADP- and ATP/ADP-Pi-actin filaments. We mapped surface-exposed cofilin-2-specific residues required for ATP-actin binding and propose that these residues function as an ‘actin nucleotide-state sensor’ among ADF/cofilins. The results suggest that cofilin-2 evolved specific biochemical and cellular properties allowing it to control actin dynamics in sarcomeres, where filament pointed ends may contain a mixture of ADP- and ATP/ADP-Pi-actin subunits. Our findings also offer a rationale for why cofilin-2 mutations in humans lead to myopathies. PMID:25373779

  19. Single-Molecule Studies of Actin Assembly and Disassembly Factors

    PubMed Central

    Smith, Benjamin A.; Gelles, Jeff; Goode, Bruce L.

    2014-01-01

    The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks. PMID:24630103

  20. Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism.

    PubMed

    Fasching, Clare L; Cejka, Petr; Kowalczykowski, Stephen C; Heyer, Wolf-Dietrich

    2015-02-19

    The displacement loop (D loop) is a DNA strand invasion product formed during homologous recombination. Disruption of nascent D loops prevents recombination, and during synthesis-dependent strand annealing (SDSA), disruption of D loops extended by DNA polymerase ensures a non-crossover outcome. The proteins implicated in D loop disruption are DNA motor proteins/helicases that act by moving DNA junctions. Here we report that D loops can also be disrupted by DNA topoisomerase 3 (Top3), and this disruption depends on Top3's catalytic activity. Yeast Top3 specifically disrupts D loops mediated by yeast Rad51/Rad54; protein-free D loops or D loop mediated by bacterial RecA protein or human RAD51/RAD54 resist dissolution. Also, the human Topoisomerase IIIa-RMI1-RMI2 complex is capable of dissolving D loops. Consistent with genetic data, we suggest that the extreme growth defect and hyper-recombination phenotype of Top3-deficient yeast cells is partially a result of unprocessed D loops. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells.

    PubMed

    Ko, Jen-Chung; Chen, Jyh-Cheng; Wang, Tai-Jing; Zheng, Hao-Yu; Chen, Wen-Ching; Chang, Po-Yuan; Lin, Yun-Wei

    2016-04-01

    Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 μM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Curcumin enhances the mitomycin C-induced cytotoxicity via downregulation of MKK1/2-ERK1/2-mediated Rad51 expression in non-small cell lung cancer cells

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

    Ko, Jen-Chung; Department of Nursing, Yuanpei University, HsinChu, Taiwan; Graduate Institute of Technology Law, National Chiao Tung University, Taiwan

    2011-09-15

    Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), has been reported to suppress the proliferation of a wide variety of tumor cells. Rad51 is a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, and HR represents a novel target for cancer therapy. A high expression of Rad51 has been reported in chemo- or radio-resistant carcinomas. Therefore, in the current study, we will examine whether curcumin could enhance the effects of mitomycin C (MMC), a DNA interstrand cross-linking agent, to induce cytotoxicity by decreasing Rad51 expression. Exposure of two human non-small lung cancer (NSCLC)more » cell lines (A549 and H1975) to curcumin could suppress MMC-induced MKK1/2-ERK1/2 signal activation and Rad51 protein expression. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased Rad51 protein levels in curcumin and MMC co-treated human lung cells. Moreover, the synergistic cytotoxic effect induced by curcumin combined with MMC was decreased by MKK1-CA-mediated enhancement of ERK1/2 activation by a significant degree. In contrast, MKK1/2 inhibitor, U0126 was shown to augment the cytotoxicity of curcumin and MMC through downregulation of ERK1/2 activation and Rad51 expression. Depletion of endogenous Rad51 expression by siRad51 RNA transfection significantly enhanced MMC and/or curcumin induced cell death and cell growth inhibition. In contrast, an overexpression of Rad51 protected lung cancer cells from synergistic cytotoxic effects induced by curcumin and MMC. We concluded that Rad51 inhibition may be an additional action mechanism for enhancing the chemosensitization of MMC by curcumin in NSCLC. - Highlights: > Curcumin downregulates MKK-ERK-mediated Rad51 expression. > Curcumin enhances mitomycin C-induced cytotoxicity. > Rad51 protects cells from cytotoxic effects induced by curcumin and mitomycin C. > Rad51 inhibition enhances the chemosensitization of

  3. Top3-Rmi1 dissolve Rad51-mediated D-loops by a topoisomerase-based mechanism

    PubMed Central

    Fasching, Clare L.; Cejka, Petr; Kowalczykowski, Stephen C.; Heyer, Wolf-Dietrich

    2015-01-01

    Summary The displacement loop (D-loop) is the DNA strand invasion product formed during homologous recombination. Disruption of nascent D-loops represents a mechanism of anti-recombination. During Synthesis-Dependent Strand Annealing D-loop disruption after extension of the invading strand is an integral step of the pathway and ensures a non-crossover outcome. The proteins implicated in D-loop disruption are DNA motor proteins/helicases acting by migrating DNA junctions. Here we report an unanticipated mechanism of D-loop dissolution mediated by DNA topoisomerase 3 (Top3) and dependent on its catalytic activity. D-loop dissolution catalyzed by yeast Top3 is highly specific for yeast Rad51/Rad54-mediated D-loops, whereas protein-free D-loops or D-loop mediated by bacterial RecA protein or human RAD51/RAD54 resist dissolution. Also the human Topoisomerase IIIα-RMI1–RMI2 complex is capable of dissolving D-loops. Consistent with genetic data, we suggest that the extreme growth defect and hyper-recombination phenotype of Top3-deficient yeast cells is in part a result of unprocessed D-loops. PMID:25699708

  4. RAD51 135G→C Modifies Breast Cancer Risk among BRCA2 Mutation Carriers: Results from a Combined Analysis of 19 Studies

    PubMed Central

    Antoniou, Antonis C. ; Sinilnikova, Olga M. ; Simard, Jacques ; Léoné, Mélanie ; Dumont, Martine ; Neuhausen, Susan L. ; Struewing, Jeffery P. ; Stoppa-Lyonnet, Dominique ; Barjhoux, Laure ; Hughes, David J. ; Coupier, Isabelle ; Belotti, Muriel ; Lasset, Christine ; Bonadona, Valérie ; Bignon, Yves-Jean ; Rebbeck, Timothy R. ; Wagner, Theresa ; Lynch, Henry T. ; Domchek, Susan M. ; Nathanson, Katherine L. ; Garber, Judy E. ; Weitzel, Jeffrey ; Narod, Steven A. ; Tomlinson, Gail ; Olopade, Olufunmilayo I. ; Godwin, Andrew ; Isaacs, Claudine ; Jakubowska, Anna ; Lubinski, Jan ; Gronwald, Jacek ; Górski, Bohdan ; Byrski, Tomasz ; Huzarski, Tomasz ; Peock, Susan ; Cook, Margaret ; Baynes, Caroline ; Murray, Alexandra ; Rogers, Mark ; Daly, Peter A. ; Dorkins, Huw ; Schmutzler, Rita K. ; Versmold, Beatrix ; Engel, Christoph ; Meindl, Alfons ; Arnold, Norbert ; Niederacher, Dieter ; Deissler, Helmut ; Spurdle, Amanda B. ; Chen, Xiaoqing ; Waddell, Nicola ; Cloonan, Nicole ; Kirchhoff, Tomas ; Offit, Kenneth ; Friedman, Eitan ; Kaufmann, Bella ; Laitman, Yael ; Galore, Gilli ; Rennert, Gad ; Lejbkowicz, Flavio ; Raskin, Leon ; Andrulis, Irene L. ; Ilyushik, Eduard ; Ozcelik, Hilmi ; Devilee, Peter ; Vreeswijk, Maaike P. G. ; Greene, Mark H. ; Prindiville, Sheila A. ; Osorio, Ana ; Benítez, Javier ; Zikan, Michal ; Szabo, Csilla I. ; Kilpivaara, Outi ; Nevanlinna, Heli ; Hamann, Ute ; Durocher, Francine ; Arason, Adalgeir ; Couch, Fergus J. ; Easton, Douglas F. ; Chenevix-Trench, Georgia 

    2007-01-01

    RAD51 is an important component of double-stranded DNA–repair mechanisms that interacts with both BRCA1 and BRCA2. A single-nucleotide polymorphism (SNP) in the 5′ untranslated region (UTR) of RAD51, 135G→C, has been suggested as a possible modifier of breast cancer risk in BRCA1 and BRCA2 mutation carriers. We pooled genotype data for 8,512 female mutation carriers from 19 studies for the RAD51 135G→C SNP. We found evidence of an increased breast cancer risk in CC homozygotes (hazard ratio [HR] 1.92 [95% confidence interval {CI} 1.25–2.94) but not in heterozygotes (HR 0.95 [95% CI 0.83–1.07]; P=.002, by heterogeneity test with 2 degrees of freedom [df]). When BRCA1 and BRCA2 mutation carriers were analyzed separately, the increased risk was statistically significant only among BRCA2 mutation carriers, in whom we observed HRs of 1.17 (95% CI 0.91–1.51) among heterozygotes and 3.18 (95% CI 1.39–7.27) among rare homozygotes (P=.0007, by heterogeneity test with 2 df). In addition, we determined that the 135G→C variant affects RAD51 splicing within the 5′ UTR. Thus, 135G→C may modify the risk of breast cancer in BRCA2 mutation carriers by altering the expression of RAD51. RAD51 is the first gene to be reliably identified as a modifier of risk among BRCA1/2 mutation carriers. PMID:17999359

  5. Meiotic Crossover Control by Concerted Action of Rad51-Dmc1 in Homolog Template Bias and Robust Homeostatic Regulation

    PubMed Central

    Huang, Chu-Chun; Grubb, Jennifer; Thacker, Drew; Lee, Chih-Ying; Dresser, Michael E.; Hunter, Neil; Bishop, Douglas K.

    2013-01-01

    During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1's strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51's strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1's chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1's dominance in promoting strand exchange between homologs involves repression of Rad51's strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to

  6. Radioresistance of chordoma cells is associated with the ATM/ATR pathway, in which RAD51 serves as an important downstream effector.

    PubMed

    Zhang, Chao; Wang, Bing; Li, Lei; Li, Yawei; Li, Pengzhi; Lv, Guohua

    2017-09-01

    Surgery followed by radiotherapy is the standard treatment for chordomas, which are a rare but low-grade type of bone cancer arising from remnants of the embryonic notochord. However, disease recurrence following radiotherapy is common, most likely due to endogenous DNA repair mechanisms that promote cell survival upon radiation strikes. The ataxia telangiectasia mutated/ataxia telangiectasia mutated and Rad3 related (ATM/ATR)-mediated pathway has a critical role in DNA repair mechanisms; however, it has rarely been investigated in chordomas. In the present study, the expression of signal molecules related to the ATM/ATR pathway in chordoma tissues and adjacent normal tissues were initially examined using immunohistochemistry and western blot analysis. Chordoma U-CH1 and U-CH2 cells were subsequently used to investigate cell responses to ionizing radiation and the potential protective actions mediated by the ATM/ATR pathway. Phosphorylated (p)-ATM, p-ATR, γ-H2A histone family, member X (H2AX) and RAD51 were significantly upregulated in chordoma tissues relative to adjacent normal tissues (P<0.05). No significant reductions were observed in the viability of U-CH1 and U-CH2 cells following exposure to low-dose (1 and 2 Gy) radiation. Radiation (1 and 2 Gy) triggered a significant upregulation in p-ATM, γ-H2AX and RAD51 expression in U-CH1 cells (P<0.05), as well as a significant upregulation in p-ATM, p-ATR and RAD51 levels in U-CH2 cells (P<0.05). RAD51 knockdown increased the responses of both U-CH1 and U-CH2 cells to 1 Gy radiation, as evidenced by the significantly decreased cell viability and increased apoptosis rate (P<0.05). Collectively, the results of the present study indicated that radioresistance of chordoma cells is associated with the ATM/ATR pathway, in which RAD51 serves as an important downstream effector. Thus, RAD51 presents a promising therapeutic target for improving the outcome of radiotherapy treatment in chordomas.

  7. Smarcal1-Mediated Fork Reversal Triggers Mre11-Dependent Degradation of Nascent DNA in the Absence of Brca2 and Stable Rad51 Nucleofilaments.

    PubMed

    Kolinjivadi, Arun Mouli; Sannino, Vincenzo; De Antoni, Anna; Zadorozhny, Karina; Kilkenny, Mairi; Técher, Hervé; Baldi, Giorgio; Shen, Rong; Ciccia, Alberto; Pellegrini, Luca; Krejci, Lumir; Costanzo, Vincenzo

    2017-09-07

    Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51 T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. Hepatocyte Growth Factor Improves the Therapeutic Efficacy of Human Bone Marrow Mesenchymal Stem Cells via RAD51.

    PubMed

    Lee, Eun Ju; Hwang, Injoo; Lee, Ji Yeon; Park, Jong Nam; Kim, Keun Cheon; Kim, Gi-Hwan; Kang, Chang-Mo; Kim, Irene; Lee, Seo-Yeon; Kim, Hyo-Soo

    2018-03-07

    Human embryonic stem cell-derived mesenchymal stem cells (hE-MSCs) have greater proliferative capacity than other human mesenchymal stem cells (hMSCs), suggesting that they may have wider applications in regenerative cellular therapy. In this study, to uncover the anti-senescence mechanism in hE-MSCs, we compared hE-MSCs with adult bone marrow (hBM-MSCs) and found that hepatocyte growth factor (HGF) was more abundantly expressed in hE-MSCs than in hBM-MSCs and that it induced the transcription of RAD51 and facilitated its SUMOylation at K70. RAD51 induction/modification by HGF not only increased telomere length but also increased mtDNA replication, leading to increased ATP generation. Moreover, HGF-treated hBM-MSCs showed significantly better therapeutic efficacy than naive hBM-MSCs. Together, the data suggest that the RAD51-mediated effects of HGF prevent hMSC senescence by promoting telomere lengthening and inducing mtDNA replication and function, which opens the prospect of developing novel therapies for liver disease. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  9. Kinetics of presynaptic filament assembly in the presence of single-stranded DNA binding protein and recombination mediator protein.

    PubMed

    Liu, Jie; Berger, Christopher L; Morrical, Scott W

    2013-11-12

    Enzymes of the RecA/Rad51 family catalyze DNA strand exchange reactions that are important for homologous recombination and for the accurate repair of DNA double-strand breaks. RecA/Rad51 recombinases are activated by their assembly into presynaptic filaments on single-stranded DNA (ssDNA), a process that is regulated by ssDNA binding protein (SSB) and mediator proteins. Mediator proteins stimulate strand exchange by accelerating the rate-limiting displacement of SSB from ssDNA by the incoming recombinase. The use of mediators is a highly conserved strategy in recombination, but the precise mechanism of mediator activity is unknown. In this study, the well-defined bacteriophage T4 recombination system (UvsX recombinase, Gp32 SSB, and UvsY mediator) is used to examine the kinetics of presynaptic filament assembly on native ssDNA in vitro. Results indicate that the ATP-dependent assembly of UvsX presynaptic filaments on Gp32-covered ssDNA is limited by a salt-sensitive nucleation step in the absence of mediator. Filament nucleation is selectively enhanced and rendered salt-resistant by mediator protein UvsY, which appears to stabilize a prenucleation complex. This mechanism potentially explains how UvsY promotes presynaptic filament assembly at physiologically relevant ionic strengths and Gp32 concentrations. Other data suggest that presynaptic filament assembly involves multiple nucleation events, resulting in many short UvsX-ssDNA filaments or clusters, which may be the relevant form for recombination in vivo. Together, these findings provide the first detailed kinetic model for presynaptic filament assembly involving all three major protein components (recombinase, mediator, and SSB) on native ssDNA.

  10. Panobinostat Enhances Cytarabine and Daunorubicin Sensitivities in AML Cells through Suppressing the Expression of BRCA1, CHK1, and Rad51

    PubMed Central

    Edwards, Holly; Caldwell, J. Timothy; Chen, Wei; Inaba, Hiroto; Xu, Xuelian; Buck, Steven A.; Taub, Jeffrey W.; Baker, Sharyn D.; Ge, Yubin

    2013-01-01

    Acute myeloid leukemia (AML) remains a challenging disease to treat and urgently requires new therapies to improve its treatment outcome. In this study, we investigated the molecular mechanisms underlying the cooperative antileukemic activities of panobinostat and cytarabine or daunorubicin (DNR) in AML cell lines and diagnostic blast samples in vitro and in vivo. Panobinostat suppressed expression of BRCA1, CHK1, and RAD51 in AML cells in a dose-dependent manner. Further, panobinostat significantly increased cytarabine- or DNR-induced DNA double-strand breaks and apoptosis, and abrogated S and/or G2/M cell cycle checkpoints. Analogous results were obtained by shRNA knockdown of BRCA1, CHK1, or RAD51. Cotreatment of NOD-SCID-IL2Rγnull mice bearing AML xenografts with panobinostat and cytarabine significantly increased survival compared to either cytarabine or panobinostat treatment alone. Additional studies revealed that panobinostat suppressed the expression of BRCA1, CHK1, and RAD51 through downregulation of E2F1 transcription factor. Our results establish a novel mechanism underlying the cooperative antileukemic activities of these drug combinations in which panobinostat suppresses expression of BRCA1, CHK1, and RAD51 to enhance cytarabine and daunorubicin sensitivities in AML cells. PMID:24244429

  11. Simultaneous ATM/BRCA1/RAD51 expression variations associated with prognostic factors in Iranian sporadic breast cancer patients.

    PubMed

    Hallajian, Zeinab; Mahjoubi, Frouzandeh; Nafissi, Nahid

    2017-07-01

    DNA double-strand breaks (DSBs) as a serious lesion are repaired by non-homologous end-joining and homologous recombination pathways. ATM, BRCA1, RAD51 genes are involved in HR pathways. While some studies have revealed individual expression changes of these genes in different types of cancer, there are limited studies attempting to evaluate correlation of expression variations of these genes in breast cancer pathogenesis. This study aimed to determine RAD51, ATM and BRCA1 gene expression level and its association with clinicopathological factors in fresh breast cancer tissues. Moreover, this study evaluates potential correlations among expression levels of these genes. 50 breast cancer tissues were collected and examined for BRCA1, RAD51 and ATM gene expression by Real Time PCR. Expression changes were analyzed with REST software version 2009. mRNA expression was reduced in all these three genes when compared with β-Actin as a control gene (P value  < 0.001). Spearman's test demonstrated a significant positive correlation among ATM, BRCA1 and RAD51 gene down expression (P value  < 0.0001). There was a significant association between down expression of ATM with stage (P value  < 0.05), necrosis (P value  < 0.05), perineural invasion (P value  < 0.05), vascular invasion (P value  < 0.01), malignancy (P value  ≤ 0.001), PR (P value  < 0.05) and ER status (P value  < 0.01). In addition, there was a significant association between down expression of BRCA1 with Ki67 (P value  ≤ 0.001). Moreover, there was a significant association between down expression of RAD51 with lymph node involvement (P value  < 0.01), auxiliary lymph node metastasis (P value  = 0.01), age (P = 0.001), grade (P value  < 0.05) and PR status (P value  < 0.05). This study suggests association between expression changes in several DSB repair genes in a common functional pathway in breast cancer and the significant association between abnormal expression of these

  12. Desmosome Assembly and Disassembly Are Membrane Raft-Dependent

    PubMed Central

    Faundez, Victor; Koval, Michael; Mattheyses, Alexa L.; Kowalczyk, Andrew P.

    2014-01-01

    Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV. PMID:24498201

  13. Recruitment of RecA homologs Dmc1p and Rad51p to the double-strand break repair site initiated by meiosis-specific endonuclease VDE (PI-SceI).

    PubMed

    Fukuda, Tomoyuki; Ohya, Yoshikazu

    2006-02-01

    During meiosis, VDE (PI-SceI), a homing endonuclease in Saccharomyces cerevisiae, introduces a double-strand break (DSB) at its recognition sequence and induces homologous recombinational repair, called homing. Meiosis-specific RecA homolog Dmc1p, as well as mitotic RecA homolog Rad51p, acts in the process of meiotic recombination, being required for strand invasion and exchange. In this study, recruitment of Dmc1p and Rad51p to the VDE-induced DSB repair site is investigated by chromatin immunoprecipitation assay. It is revealed that Dmc1p and Rad51p are loaded to the repair site in an independent manner. Association of Rad51p requires other DSB repair proteins of Rad52p, Rad55p, and Rad57p, while loading of Dmc1p is facilitated by the different protein, Sae3p. Absence of Tid1p, which can bind both RecA homologs, appears specifically to cause an abnormal distribution of Dmc1p. Lack of Hop2, Mnd1p, and Sae1p does not impair recruitment of both RecA homologs. These findings reveal the discrete functions of each strand invasion protein in VDE-initiated homing, confirm the similarity between VDE-initiated homing and Spo11p-initiated meiotic recombination, and demonstrate the availability of VDE-initiated homing for the study of meiotic recombination.

  14. Enhanced Histone Deacetylase Activity in Malignant Melanoma Provokes RAD51 and FANCD2-Triggered Drug Resistance.

    PubMed

    Krumm, Andrea; Barckhausen, Christina; Kücük, Pelin; Tomaszowski, Karl-Heinz; Loquai, Carmen; Fahrer, Jörg; Krämer, Oliver Holger; Kaina, Bernd; Roos, Wynand Paul

    2016-05-15

    DNA-damaging anticancer drugs remain a part of metastatic melanoma therapy. Epigenetic reprogramming caused by increased histone deacetylase (HDAC) activity arising during tumor formation may contribute to resistance of melanomas to the alkylating drugs temozolomide, dacarbazine, and fotemustine. Here, we report on the impact of class I HDACs on the response of malignant melanoma cells treated with alkylating agents. The data show that malignant melanomas in situ contain a high level of HDAC1/2 and malignant melanoma cells overexpress HDAC1/2/3 compared with noncancer cells. Furthermore, pharmacologic inhibition of class I HDACs sensitizes malignant melanoma cells to apoptosis following exposure to alkylating agents, while not affecting primary melanocytes. Inhibition of HDAC1/2/3 caused sensitization of melanoma cells to temozolomide in vitro and in melanoma xenografts in vivo HDAC1/2/3 inhibition resulted in suppression of DNA double-strand break (DSB) repair by homologous recombination because of downregulation of RAD51 and FANCD2. This sensitized cells to the cytotoxic DNA lesion O(6)-methylguanine and caused a synthetic lethal interaction with the PARP-1 inhibitor olaparib. Furthermore, knockdown experiments identified HDAC2 as being responsible for the regulation of RAD51. The influence of class I HDACs on DSB repair by homologous recombination and the possible clinical implication on malignant melanoma therapy with temozolomide and other alkylating drugs suggests a combination approach where class I HDAC inhibitors such as valproic acid or MS-275 (entinostat) appear to counteract HDAC- and RAD51/FANCD2-mediated melanoma cell resistance. Cancer Res; 76(10); 3067-77. ©2016 AACR. ©2016 American Association for Cancer Research.

  15. Dose-Response Curves of the FDXR and RAD51 Genes with 6 and 18 MV Beam Energies in Human Peripheral Blood Lymphocytes.

    PubMed

    Saberi, Alihossein; Khodamoradi, Ehsan; Tahmasebi Birgani, Mohammad Javad; Makvandi, Manoochehr; Noori, Bijan

    2016-11-01

    Rapid dose assessment using biological dosimetry methods is essential to increase the chance of survival of exposed individuals in radiation accidents. We compared the expression levels of the FDXR and RAD51 genes at 6 and 18 MV beam energies in human peripheral blood lymphocytes. The results of our study can be used to analyze radiation energy in biological dosimetry. For this in vitro experimental study, from 36 students in the medical physics and virology departments, seven voluntary, healthy, non-smoking male blood donors of Khuzestan ethnicity with no history of exposure to ionization radiation were selected using simple randomized sampling. Sixty-three peripheral blood samples were collected from the seven healthy donors. Human peripheral blood was then exposed to doses of 0, 0.2, 0.5, 2, and 4 Gy with 6 and 18 MV beam energies in a Linac Varian 2100C/D (Varian, USA) at Golestan hospital in Ahvaz, Iran. After RNA extraction and cDNA synthesis, the expression levels of FDXR and RAD51 were determined 24 hours post-irradiation using the gel-purified reverse transcription polymerase chain reaction (RT-PCR) technique and TaqMan strategy (by real-time PCR). The expression level of FDXR gene was significantly increased at doses of 2 Gy and 4 Gy in the 6 - 18 MV energy range (P < 0.001 and P < 0.008, respectively). The medians with interquartile ranges (IQRs) of the copy numbers of the FDXR gene at 2 Gy and 4 Gy doses under 6 and 18 MV beam energies were 2393.59 (1798.21, 2575.37) and 2983.00 (2199.48, 3643.82) and 3779.12 (3051.40, 5120.74) and 5051.26 (4704.83, 5859.17), respectively. However, RAD51 gene expression levels only showed a significant difference between samples at a dose of 2 Gy with 6 and 18 MV beam energies, respectively (P < 0.040). The medians with IQRs of the copy numbers of the RAD51 gene were 2092.77 (1535.78, 2705.61) and 3412.57 (2979.72, 4530.61) at beam energies of 6 and 18 MV, respectively. The data suggest that the expression analysis of

  16. Rad52/Rad59-dependent Recombination as a Means to Rectify Faulty Okazaki Fragment Processing*

    PubMed Central

    Lee, Miju; Lee, Chul-Hwan; Demin, Annie Albert; Munashingha, Palinda Ruvan; Amangyeld, Tamir; Kwon, Buki; Formosa, Tim; Seo, Yeon-Soo

    2014-01-01

    The correct removal of 5′-flap structures by Rad27 and Dna2 during Okazaki fragment maturation is crucial for the stable maintenance of genetic materials and cell viability. In this study, we identified RAD52, a key recombination protein, as a multicopy suppressor of dna2-K1080E, a lethal helicase-negative mutant allele of DNA2 in yeasts. In contrast, the overexpression of Rad51, which works conjointly with Rad52 in canonical homologous recombination, failed to suppress the growth defect of the dna2-K1080E mutation, indicating that Rad52 plays a unique and distinct role in Okazaki fragment metabolism. We found that the recombination-defective Rad52-QDDD/AAAA mutant did not rescue dna2-K1080E, suggesting that Rad52-mediated recombination is important for suppression. The Rad52-mediated enzymatic stimulation of Dna2 or Rad27 is not a direct cause of suppression observed in vivo, as both Rad52 and Rad52-QDDD/AAAA proteins stimulated the endonuclease activities of both Dna2 and Rad27 to a similar extent. The recombination mediator activity of Rad52 was dispensable for the suppression, whereas both the DNA annealing activity and its ability to interact with Rad59 were essential. In addition, we found that several cohesion establishment factors, including Rsc2 and Elg1, were required for the Rad52-dependent suppression of dna2-K1080E. Our findings suggest a novel Rad52/Rad59-dependent, but Rad51-independent recombination pathway that could ultimately lead to the removal of faulty flaps in conjunction with cohesion establishment factors. PMID:24711454

  17. Elastic deformation and failure in protein filament bundles: Atomistic simulations and coarse-grained modeling.

    PubMed

    Hammond, Nathan A; Kamm, Roger D

    2008-07-01

    The synthetic peptide RAD16-II has shown promise in tissue engineering and drug delivery. It has been studied as a vehicle for cell delivery and controlled release of IGF-1 to repair infarcted cardiac tissue, and as a scaffold to promote capillary formation for an in vitro model of angiogenesis. The structure of RAD16-II is hierarchical, with monomers forming long beta-sheets that pair together to form filaments; filaments form bundles approximately 30-60 nm in diameter; branching networks of filament bundles form macroscopic gels. We investigate the mechanics of shearing between the two beta-sheets constituting one filament, and between cohered filaments of RAD16-II. This shear loading is found in filament bundle bending or in tensile loading of fibers composed of partial-length filaments. Molecular dynamics simulations show that time to failure is a stochastic function of applied shear stress, and that for a given loading time behavior is elastic for sufficiently small shear loads. We propose a coarse-grained model based on Langevin dynamics that matches molecular dynamics results and facilities extending simulations in space and time. The model treats a filament as an elastic string of particles, each having potential energy that is a periodic function of its position relative to the neighboring filament. With insight from these simulations, we discuss strategies for strengthening RAD16-II and similar materials.

  18. Cloning, sequencing, disruption and phenotypic analysis of uvsC, an Aspergillus nidulans homologue of yeast RAD51.

    PubMed

    van Heemst, D; Swart, K; Holub, E F; van Dijk, R; Offenberg, H H; Goosen, T; van den Broek, H W; Heyting, C

    1997-05-01

    We have cloned the uvsC gene of Aspergillus nidulans by complementation of the A. nidulans uvsC114 mutant. The predicted protein UVSC shows 67.4% sequence identity to the Saccharomyces cerevisiae Rad51 protein and 27.4% sequence identity to the Escherichia coli RecA protein. Transcription of uvsC is induced by methyl-methane sulphonate (MMS), as is transcription of RAD51 of yeast. Similar levels of uvsC transcription were observed after MMS induction in a uvsC+ strain and the uvsC114 mutant. The coding sequence of the uvsC114 allele has a deletion of 6 bp, which results in deletion of two amino acids and replacement of one amino acid in the translation product. In order to gain more insight into the biological function of the uvsC gene, a uvsC null mutant was constructed, in which the entire uvsC coding sequence was replaced by a selectable marker gene. Meiotic and mitotic phenotypes of a uvsC+ strain, the uvsC114 mutant and the uvsC null mutant were compared. The uvsC null mutant was more sensitive to both UV and MMS than the uvsC114 mutant. The uvsC114 mutant arrested in meiotic prophase-I. The uvsC null mutant arrested at an earlier stage, before the onset of meiosis. One possible interpretation of these meiotic phenotypes is that the A. nidulans homologue of Rad51 of yeast has a role both in the specialized processes preceding meiosis and in meiotic prophase I.

  19. Functional conservation of the yeast and Arabidopsis RAD54-like genes.

    PubMed

    Klutstein, Michael; Shaked, Hezi; Sherman, Amir; Avivi-Ragolsky, Naomi; Shema, Efrat; Zenvirth, Drora; Levy, Avraham A; Simchen, Giora

    2008-04-01

    The Saccharomyces cerevisiae RAD54 gene has critical roles in DNA double-strand break repair, homologous recombination, and gene targeting. Previous results show that the yeast gene enhances gene targeting when expressed in Arabidopsis thaliana. In this work we address the trans-species compatibility of Rad54 functions. We show that overexpression of yeast RAD54 in Arabidopsis enhances DNA damage resistance severalfold. Thus, the yeast gene is active in the Arabidopsis homologous-recombination repair system. Moreover, we have identified an A. thaliana ortholog of yeast RAD54, named AtRAD54. This gene, with close sequence similarity to RAD54, complements methylmethane sulfonate (MMS) sensitivity but not UV sensitivity or gene targeting defects of rad54Delta mutant yeast cells. Overexpression of AtRAD54 in Arabidopsis leads to enhanced resistance to DNA damage. This gene's assignment as a RAD54 ortholog is further supported by the interaction of AtRad54 with AtRad51 and the interactions between alien proteins (i.e., yeast Rad54 with AtRAD51 and yeast Rad51 with AtRad54) in a yeast two-hybrid experiment. These interactions hint at the molecular nature of this interkingdom complementation, although the stronger effect of the yeast Rad54 in plants than AtRad54 in yeast might be explained by an ability of the Rad54 protein to act alone, independently of its interaction with Rad51.

  20. A disassembly-driven mechanism explains F-actin-mediated chromosome transport in starfish oocytes

    PubMed Central

    Bun, Philippe; Dmitrieff, Serge; Belmonte, Julio M

    2018-01-01

    While contraction of sarcomeric actomyosin assemblies is well understood, this is not the case for disordered networks of actin filaments (F-actin) driving diverse essential processes in animal cells. For example, at the onset of meiosis in starfish oocytes a contractile F-actin network forms in the nuclear region transporting embedded chromosomes to the assembling microtubule spindle. Here, we addressed the mechanism driving contraction of this 3D disordered F-actin network by comparing quantitative observations to computational models. We analyzed 3D chromosome trajectories and imaged filament dynamics to monitor network behavior under various physical and chemical perturbations. We found no evidence of myosin activity driving network contractility. Instead, our observations are well explained by models based on a disassembly-driven contractile mechanism. We reconstitute this disassembly-based contractile system in silico revealing a simple architecture that robustly drives chromosome transport to prevent aneuploidy in the large oocyte, a prerequisite for normal embryonic development. PMID:29350616

  1. Nano-functionalized filamentous fungus hyphae with fast reversible macroscopic assembly & disassembly features.

    PubMed

    Wang, Haiying; Li, Xiaorui; Chai, Liyuan; Zhang, Liyuan

    2015-05-18

    A uniform decoration of hyphae by polyaniline nanoparticles (PANI NPs) was achieved here. This novel hybrid structure can be effectively assembled into a film by filtration and disassembled in water by shaking. This reversible process is very fast, which promises applications in nanomaterials including adsorption.

  2. Homologous Recombination Repair Signaling in Chemical Carcinogenesis: Prolonged Particulate Hexavalent Chromium Exposure Suppresses the Rad51 Response in Human Lung Cells

    PubMed Central

    Qin, Qin; Xie, Hong; Wise, Sandra S.; Browning, Cynthia L.; Thompson, Kelsey N.; Holmes, Amie L.; Wise, John Pierce

    2014-01-01

    The aim of this study was to focus on hexavalent chromium, [Cr(VI)], a chemical carcinogen and major public health concern, and consider its ability to impact DNA double strand break repair. We further focused on particulate Cr(VI), because it is the more potent carcinogenic form of Cr(VI). DNA double strand break repair serves to protect cells against the detrimental effects of DNA double strand breaks. For particulate Cr(VI), data show DNA double strand break repair must be overcome for neoplastic transformation to occur. Acute Cr(VI) exposures reveal a robust DNA double strand break repair response, however, longer exposures have not been considered. Using the comet assay, we found longer exposures to particulate zinc chromate induced concentration-dependent increases in DNA double strand breaks indicating breaks were occurring throughout the exposure time. Acute (24 h) exposure induced DNA double strand break repair signaling by inducing Mre11 foci formation, ATM phosphorylation and phosphorylated ATM foci formation, Rad51 protein levels and Rad51 foci formation. However, longer exposures reduced the Rad51 response. These data indicate a major chemical carcinogen can simultaneously induce DNA double strand breaks and alter their repair and describe a new and important aspect of the carcinogenic mechanism for Cr(VI). PMID:25173789

  3. BRCA2 and RAD51 promote double-strand break formation and cell death in response to gemcitabine.

    PubMed

    Jones, Rebecca M; Kotsantis, Panagiotis; Stewart, Grant S; Groth, Petra; Petermann, Eva

    2014-10-01

    Replication inhibitors cause replication fork stalling and double-strand breaks (DSB) that result from processing of stalled forks. During recovery from replication blocks, the homologous recombination (HR) factor RAD51 mediates fork restart and DSB repair. HR defects therefore sensitize cells to replication inhibitors, with clear implications for cancer therapy. Gemcitabine is a potent replication inhibitor used to treat cancers with mutations in HR genes such as BRCA2. Here, we investigate why, paradoxically, mutations in HR genes protect cells from killing by gemcitabine. Using DNA replication and DNA damage assays in mammalian cells, we show that even short gemcitabine treatments cause persistent replication inhibition. BRCA2 and RAD51 are recruited to chromatin early after removal of the drug, actively inhibit replication fork progression, and promote the formation of MUS81- and XPF-dependent DSBs that remain unrepaired. Our data suggest that HR intermediates formed at gemcitabine-stalled forks are converted into DSBs and thus contribute to gemcitabine-induced cell death, which could have implications for the treatment response of HR-deficient tumors. ©2014 American Association for Cancer Research.

  4. BRCA2 and RAD51 promote double-strand break formation and cell death in response to Gemcitabine

    PubMed Central

    Jones, Rebecca M.; Kotsantis, Panagiotis; Stewart, Grant S.; Groth, Petra; Petermann, Eva

    2014-01-01

    Replication inhibitors cause replication fork stalling and double-strand breaks (DSBs) that result from processing of stalled forks. During recovery from replication blocks, the homologous recombination (HR) factor RAD51 mediates fork restart and DSB repair. HR defects therefore sensitise cells to replication inhibitors, with clear implications for cancer therapy. Gemcitabine is a potent replication inhibitor used to treat cancers with mutations in HR genes such as BRCA2. Here we investigate why, paradoxically, mutations in HR genes protect cells from killing by Gemcitabine. Using DNA replication and -damage assays in mammalian cells, we show that even short Gemcitabine treatments cause persistent replication inhibition. BRCA2 and RAD51 are recruited to chromatin early after removal of the drug, actively inhibit replication fork progression and promote the formation of MUS81- and XPF-dependent DSBs that remain unrepaired. Our data suggest that HR intermediates formed at Gemcitabine-stalled forks are converted into DSBs and thus contribute to Gemcitabine-induced cell death, which could have implications for the treatment response of HR-deficient tumours. PMID:25053826

  5. The fission yeast meiosis-specific Dmc1 recombinase mediates formation and branch migration of Holliday junctions by preferentially promoting strand exchange in a direction opposite to that of Rad51

    PubMed Central

    Murayama, Yasuto; Tsutsui, Yasuhiro; Iwasaki, Hiroshi

    2011-01-01

    Homologous recombination proceeds via the formation of several intermediates including Holliday junctions (HJs), which are important for creating crossover products. DNA strand exchange is a core reaction that produces these intermediates that is directly catalyzed by RecA family recombinases, of which there are two types in eukaryotes: universal Rad51 and meiosis-specific Dmc1. We demonstrated previously that Rad51 promotes four-strand exchange, mimicking the formation and branch migration of HJs. Here we show that Dmc1 from fission yeast has a similar activity, which requires ATP hydrolysis and is independent of an absolute requirement for the Swi5–Sfr1 complex. These features are critically different from three-strand exchange mediated by Dmc1, but similar to those of four-strand exchange mediated by Rad51, suggesting that strand exchange reactions between duplex–duplex and single-duplex DNAs are mechanistically different. Interestingly, despite similarities in protein structure and in reaction features, the preferential polarities of Dmc1 and Rad51 strand exchange are different (Dmc1 promotes exchange in the 5′-to-3′ direction and Rad51 promotes exchange in the 3′-to-5′ direction relative to the ssDNA region of the DNA substrate). The significance of the Dmc1 polarity is discussed within the context of the necessity for crossover production. PMID:21363965

  6. RECK impedes DNA repair by inhibiting the erbB/JAB1/Rad51 signaling axis and enhances chemosensitivity of breast cancer cells

    PubMed Central

    Hong, Kun-Jing; Hsu, Ming-Chuan; Hung, Wen-Chun

    2015-01-01

    The reversion-inducing cysteine-rich protein with kazal motif (RECK) is an endogenous matrix metalloproteinase (MMP) inhibitor and a tumor suppressor. Its expression is dramatically down-regulated in human cancers. Our recent results suggest a novel MMP-independent anti-cancer activity of RECK by inhibiting the erbB signaling. Activation of the erbB signaling is associated with chemotherapeutic resistance, however, whether RECK could modulate drug sensitivity is still unknown. Here we demonstrated that expression of RECK induced the activation of ATM and ATR pathways, and the formation of γ-H2AX foci in breast cancer cells. RECK inhibited the erbB signaling and attenuated the expression of the downstream molecules Jun activation domain-binding protein 1 (JAB1) and the DNA repair protein RAD51 to impede DNA repair and to increase drug sensitivity. Treatment of epidermal growth factor or over-expression of HER-2 effectively reversed the inhibitory effect of RECK. In addition, ectopic expression of JAB1 counteracted RECK-induced RAD51 reduction and drug sensitization. Our results elucidate a novel function of RECK to modulate DNA damage response and drug resistance by inhibiting the erbB/Jab1/RAD51 signaling axis. Restoration of RECK expression in breast cancer cells may increase sensitivity to chemotherapeutic agents. PMID:26396917

  7. Identification of the meiotic toolkit in diatoms and exploration of meiosis-specific SPO11 and RAD51 homologs in the sexual species Pseudo-nitzschia multistriata and Seminavis robusta.

    PubMed

    Patil, Shrikant; Moeys, Sara; von Dassow, Peter; Huysman, Marie J J; Mapleson, Daniel; De Veylder, Lieven; Sanges, Remo; Vyverman, Wim; Montresor, Marina; Ferrante, Maria Immacolata

    2015-11-14

    Sexual reproduction is an obligate phase in the life cycle of most eukaryotes. Meiosis varies among organisms, which is reflected by the variability of the gene set associated to the process. Diatoms are unicellular organisms that belong to the stramenopile clade and have unique life cycles that can include a sexual phase. The exploration of five diatom genomes and one diatom transcriptome led to the identification of 42 genes potentially involved in meiosis. While these include the majority of known meiosis-related genes, several meiosis-specific genes, including DMC1, could not be identified. Furthermore, phylogenetic analyses supported gene identification and revealed ancestral loss and recent expansion in the RAD51 family in diatoms. The two sexual species Pseudo-nitzschia multistriata and Seminavis robusta were used to explore the expression of meiosis-related genes: RAD21, SPO11-2, RAD51-A, RAD51-B and RAD51-C were upregulated during meiosis, whereas other paralogs in these families showed no differential expression patterns, suggesting that they may play a role during vegetative divisions. An almost identical toolkit is shared among Pseudo-nitzschia multiseries and Fragilariopsis cylindrus, as well as two species for which sex has not been observed, Phaeodactylum tricornutum and Thalassiosira pseudonana, suggesting that these two may retain a facultative sexual phase. Our results reveal the conserved meiotic toolkit in six diatom species and indicate that Stramenopiles share major modifications of canonical meiosis processes ancestral to eukaryotes, with important divergences in each Kingdom.

  8. Presynaptic Filament Dynamics in Homologous Recombination and DNA Repair

    PubMed Central

    Liu, Jie; Ehmsen, Kirk T.; Heyer, Wolf-Dietrich; Morrical, Scott W.

    2014-01-01

    Homologous Recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments—helical filaments of a recombinase enzyme bound to single-stranded DNA. Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we review the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments: some intrinsic such as recombinase ATP binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examine dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examine the biochemical properties of recombination proteins from four model systems (T4 phage, E. coli, S. cerevisiae, and H. sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We propose that the presynaptic filament has evolved to rely on multiple external factors for increased multi-level regulation of HR processes in genomes with greater structural and sequence complexity. PMID:21599536

  9. Vital Roles of the Second DNA-binding Site of Rad52 Protein in Yeast Homologous Recombination*

    PubMed Central

    Arai, Naoto; Kagawa, Wataru; Saito, Kengo; Shingu, Yoshinori; Mikawa, Tsutomu; Kurumizaka, Hitoshi; Shibata, Takehiko

    2011-01-01

    RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a “recombination mediator” to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing. PMID:21454474

  10. Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

    PubMed Central

    Malivert, Laurent; McIlwraith, Michael J.; Pape, Tillman; West, Stephen C.; Zhang, Xiaodong

    2014-01-01

    Mutations in BRCA2 increase susceptibility to breast, ovarian and prostate cancers. The product of human BRCA2, BRCA2 protein, plays a key role in the repair of DNA double strand breaks and interstrand crosslinks by RAD51-mediated homologous recombination. Here, we present a biochemical and structural characterization of full length (3,418 amino acid) BRCA2, alone and in complex with RAD51. We show that BRCA2 facilitates nucleation of RAD51 filaments at multiple sites on single-stranded DNA. Three-dimensional electron microscopy reconstructions revealed that BRCA2 exists as a dimer and that two oppositely-oriented sets of RAD51 molecules bind the dimer. Single stranded DNA binds along the long axis of BRCA2, such that only one set of RAD51 monomers can form a productive complex with DNA and establish filament formation. Our data define the molecular mechanism by which this tumor suppressor facilitates RAD51-mediated homologous recombinational repair. PMID:25282148

  11. Corylin increases the sensitivity of hepatocellular carcinoma cells to chemotherapy through long noncoding RNA RAD51-AS1-mediated inhibition of DNA repair.

    PubMed

    Chen, Chin-Chuan; Chen, Chi-Yuan; Ueng, Shir-Hwa; Hsueh, Chuen; Yeh, Chau-Ting; Ho, Jar-Yi; Chou, Li-Fang; Wang, Tong-Hong

    2018-05-10

    Corylin, a biologically active agent extracted from Psoralea corylifolia L. (Fabaceae), promotes bone differentiation and inhibits inflammation. Currently, few reports have addressed the biological functions that are regulated by corylin, and to date, no studies have investigated its antitumor activity. In this study, we used cell functional assays to analyze the antitumor activity of corylin in hepatocellular carcinoma (HCC). Furthermore, whole-transcriptome assays were performed to identify the downstream genes that were regulated by corylin, and gain-of-function and loss-of-function experiments were conducted to examine the regulatory roles of the above genes. We found that corylin significantly inhibited the proliferation, migration, and invasion of HCC cells and increased the toxic effects of chemotherapeutic agents against HCC cells. These properties were due to the induction of a long noncoding RNA, RAD51-AS1, which bound to RAD51 mRNA, thereby inhibiting RAD51 protein expression, thus inhibiting the DNA damage repair ability of HCC cells. Animal experiments also showed that a combination treatment with corylin significantly increased the inhibitory effects of the chemotherapeutic agent etoposide (VP16) on tumor growth. These findings indicate that corylin has strong potential as an adjuvant drug in HCC treatment and that corylin can strengthen the therapeutic efficacy of chemotherapy and radiotherapy.

  12. Identification of the meiotic toolkit in diatoms and exploration of meiosis-specific SPO11 and RAD51 homologs in the sexual species Pseudo-nitzschia multistriata and Seminavis robusta

    DOE PAGES

    Patil, Shrikant; Moeys, Sara; von Dassow, Peter; ...

    2015-11-14

    Sexual reproduction is an obligate phase in the life cycle of most eukaryotes. Meiosis varies among organisms, which is reflected by the variability of the gene set associated to the process. Diatoms are unicellular organisms that belong to the stramenopile clade and have unique life cycles that can include a sexual phase. The exploration of five diatom genomes and one diatom transcriptome led to the identification of 42 genes potentially involved in meiosis. While these include the majority of known meiosis-related genes, several meiosis-specific genes, including DMC1, could not be identified. Furthermore, phylogenetic analyses supported gene identification and revealed ancestralmore » loss and recent expansion in the RAD51 family in diatoms. The two sexual species Pseudo-nitzschia multistriata and Seminavis robusta were used to explore the expression of meiosis-related genes: RAD21, SPO11-2, RAD51-A, RAD51-B and RAD51-C were upregulated during meiosis, whereas other paralogs in these families showed no differential expression patterns, suggesting that they may play a role during vegetative divisions. An almost identical toolkit is shared among Pseudo-nitzschia multiseries and Fragilariopsis cylindrus, as well as two species for which sex has not been observed, Phaeodactylum tricornutum and Thalassiosira pseudonana, suggesting that these two may retain a facultative sexual phase. Lastly, our results reveal the conserved meiotic toolkit in six diatom species and indicate that Stramenopiles share major modifications of canonical meiosis processes ancestral to eukaryotes, with important divergences in each Kingdom.« less

  13. Identification of the meiotic toolkit in diatoms and exploration of meiosis-specific SPO11 and RAD51 homologs in the sexual species Pseudo-nitzschia multistriata and Seminavis robusta

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

    Patil, Shrikant; Moeys, Sara; von Dassow, Peter

    Sexual reproduction is an obligate phase in the life cycle of most eukaryotes. Meiosis varies among organisms, which is reflected by the variability of the gene set associated to the process. Diatoms are unicellular organisms that belong to the stramenopile clade and have unique life cycles that can include a sexual phase. The exploration of five diatom genomes and one diatom transcriptome led to the identification of 42 genes potentially involved in meiosis. While these include the majority of known meiosis-related genes, several meiosis-specific genes, including DMC1, could not be identified. Furthermore, phylogenetic analyses supported gene identification and revealed ancestralmore » loss and recent expansion in the RAD51 family in diatoms. The two sexual species Pseudo-nitzschia multistriata and Seminavis robusta were used to explore the expression of meiosis-related genes: RAD21, SPO11-2, RAD51-A, RAD51-B and RAD51-C were upregulated during meiosis, whereas other paralogs in these families showed no differential expression patterns, suggesting that they may play a role during vegetative divisions. An almost identical toolkit is shared among Pseudo-nitzschia multiseries and Fragilariopsis cylindrus, as well as two species for which sex has not been observed, Phaeodactylum tricornutum and Thalassiosira pseudonana, suggesting that these two may retain a facultative sexual phase. Lastly, our results reveal the conserved meiotic toolkit in six diatom species and indicate that Stramenopiles share major modifications of canonical meiosis processes ancestral to eukaryotes, with important divergences in each Kingdom.« less

  14. Disassemblability modeling technology of configurable product based on disassembly constraint relation weighted design structure matrix(DSM)

    NASA Astrophysics Data System (ADS)

    Qiu, Lemiao; Liu, Xiaojian; Zhang, Shuyou; Sun, Liangfeng

    2014-05-01

    The current research of configurable product disassemblability focuses on disassemblability evaluation and disassembly sequence planning. Little work has been done on quantitative analysis of configurable product disassemblability. The disassemblability modeling technology for configurable product based on disassembly constraint relation weighted design structure matrix (DSM) is proposed. Major factors affecting the disassemblability of configurable product are analyzed, and the disassembling degrees between components in configurable product are obtained by calculating disassembly entropies such as joint type, joint quantity, disassembly path, disassembly accessibility and material compatibility. The disassembly constraint relation weighted DSM of configurable product is constructed and configuration modules are formed by matrix decomposition and tearing operations. The disassembly constraint relation in configuration modules is strong coupling, and the disassembly constraint relation between modules is weak coupling, and the disassemblability configuration model is constructed based on configuration module. Finally, taking a hydraulic forging press as an example, the decomposed weak coupling components are used as configuration modules alone, components with a strong coupling are aggregated into configuration modules, and the disassembly sequence of components inside configuration modules is optimized by tearing operation. A disassemblability configuration model of the hydraulic forging press is constructed. By researching the disassemblability modeling technology of product configuration design based on disassembly constraint relation weighted DSM, the disassembly property in maintenance, recycling and reuse of configurable product are optimized.

  15. Interactions of RadB, a DNA repair protein in archaea, with DNA and ATP.

    PubMed

    Guy, Colin P; Haldenby, Sam; Brindley, Amanda; Walsh, David A; Briggs, Geoffrey S; Warren, Martin J; Allers, Thorsten; Bolt, Edward L

    2006-04-21

    The RecA family of recombinases (RecA, Rad51, RadA and UvsX) catalyse strand-exchange between homologous DNA molecules by utilising conserved DNA-binding modules and a common core ATPase domain. RadB was identified in archaea as a Rad51-like protein on the basis of conserved ATPase sequences. However, RadB does not catalyse strand exchange and does not turn over ATP efficiently. RadB does bind DNA, and here we report a triplet of residues (Lys-His-Arg) that is highly conserved at the RadB C terminus, and is crucial for DNA binding. This is consistent with the motif forming a "basic patch" of highly conserved residues identified in an atomic structure of RadB from Thermococcus kodakaraensis. As the triplet motif is conserved at the C terminus of XRCC2 also, a mammalian Rad51-paralogue, we present a phylogenetic analysis that clarifies the relationship between RadB, Rad51-paralogues and recombinases. We investigate interactions between RadB and ATP using genetics and biochemistry; ATP binding by RadB is needed to promote survival of Haloferax volcanii after UV irradiation, and ATP, but not other NTPs, induces pronounced conformational change in RadB. This is the first genetic analysis of radB, and establishes its importance for maintaining genome stability in archaea. ATP-induced conformational change in RadB may explain previous reports that RadB controls Holliday junction resolution by Hjc, depending on the presence or the absence of ATP.

  16. Recruitment Kinetics of Tropomyosin Tpm3.1 to Actin Filament Bundles in the Cytoskeleton Is Independent of Actin Filament Kinetics.

    PubMed

    Appaduray, Mark A; Masedunskas, Andrius; Bryce, Nicole S; Lucas, Christine A; Warren, Sean C; Timpson, Paul; Stear, Jeffrey H; Gunning, Peter W; Hardeman, Edna C

    2016-01-01

    The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.

  17. Exact Length Distribution of Filamentous Structures Assembled from a Finite Pool of Subunits.

    PubMed

    Harbage, David; Kondev, Jané

    2016-07-07

    Self-assembling filamentous structures made of protein subunits are ubiquitous in cell biology. These structures are often highly dynamic, with subunits in a continuous state of flux, binding to and falling off of filaments. In spite of this constant turnover of their molecular parts, many cellular structures seem to maintain a well-defined size over time, which is often required for their proper functioning. One widely discussed mechanism of size regulation involves the cell maintaining a finite pool of protein subunits available for assembly. This finite pool mechanism can control the length of a single filament by having assembly proceed until the pool of free subunits is depleted to the point when assembly and disassembly are balanced. Still, this leaves open the question of whether the same mechanism can provide size control for multiple filamentous structures that are assembled from a common pool of protein subunits, as is often the case in cells. We address this question by solving the steady-state master equation governing the stochastic assembly and disassembly of multifilament structures made from a shared finite pool of subunits. We find that, while the total number of subunits within a multifilament structure is well-defined, individual filaments within the structure have a wide, power-law distribution of lengths. We also compute the phase diagram for two multifilament structures competing for the same pool of subunits and identify conditions for coexistence when both have a well-defined size. These predictions can be tested in cell experiments in which the size of the subunit pool or the number of filament nucleators is tuned.

  18. Long-range self-organization of cytoskeletal myosin II filament stacks.

    PubMed

    Hu, Shiqiong; Dasbiswas, Kinjal; Guo, Zhenhuan; Tee, Yee-Han; Thiagarajan, Visalatchi; Hersen, Pascal; Chew, Teng-Leong; Safran, Samuel A; Zaidel-Bar, Ronen; Bershadsky, Alexander D

    2017-02-01

    Although myosin II filaments are known to exist in non-muscle cells, their dynamics and organization are incompletely understood. Here, we combined structured illumination microscopy with pharmacological and genetic perturbations, to study the process of actomyosin cytoskeleton self-organization into arcs and stress fibres. A striking feature of the myosin II filament organization was their 'registered' alignment into stacks, spanning up to several micrometres in the direction orthogonal to the parallel actin bundles. While turnover of individual myosin II filaments was fast (characteristic half-life time 60 s) and independent of actin filament turnover, the process of stack formation lasted a longer time (in the range of several minutes) and required myosin II contractility, as well as actin filament assembly/disassembly and crosslinking (dependent on formin Fmnl3, cofilin1 and α-actinin-4). Furthermore, myosin filament stack formation involved long-range movements of individual myosin filaments towards each other suggesting the existence of attractive forces between myosin II filaments. These forces, possibly transmitted via mechanical deformations of the intervening actin filament network, may in turn remodel the actomyosin cytoskeleton and drive its self-organization.

  19. Estrogen Modulation of MgATPase Activity of Nonmuscle Myosin-II-B Filaments

    PubMed Central

    Gorodeski, George I.

    2008-01-01

    The study tested the hypothesis that estrogen controls epithelial paracellular resistance through modulation of myosin. The objective was to understand how estrogen modulates non-muscle myosin-II-B (NMM-II-B), the main component of the cortical actomyosin in human epithelial cervical cells. Experiments used human cervical epithelial cells CaSki as a model, and end points were NMM-II-B phosphorylation, filamentation, and MgATPase activity. The results were as follows: 1) treatment with estrogen increased phosphorylation and MgATPase activity and decreased NMM-II-B filamentation; 2) estrogen effects could be blocked by antisense nucleotides for the estrogen receptor-α and by ICI-182,780, tamoxifen, and the casein kinase-II (CK2) inhibitor, 5,6-dichloro-1-β-(D)-ribofuranosylbenzimidazole and attenuated by AG1478 and PD98059 (inhibitors of epithelial growth factor receptor and ERK/MAPK) but not staurosporine [blocker of protein kinase C (PKC)]; 3) treatments with the PKC activator sn-1,2-di-octanoyl diglyceride induced biphasic effect on NMM-II-B MgATPase activity: an increase at 1 nM to 1 μM and a decrease in activity at more than 1 μM; 4) sn-1,2-dioctanoyl diglyceride also decreased NMM-II-B filamentation in a monophasic and saturable dose dependence (EC50 1–10 μM); 5) when coincubated directly with purified NMM-II-B filaments, both CK2 and PKC decreased filamentation and increased MgATPase activity; 6) assays done on disassembled NMM-II-B filaments showed MgATPase activity in filaments obtained from estrogen-treated cells but not estrogen-depleted cells; and 7) incubations in vitro with CK2, but not PKC, facilitated MgATPase activity, even in disassembled NMM-II-B filaments. The results suggest that estrogen, in an effect mediated by estrogen receptor-α and CK2 and involving the epithelial growth factor receptor and ERK/MAPK cascades, increases NMM-II-B MgATPase activity independent of NMM-II-B filamentation status. PMID:17023528

  20. Role of allosteric switch residue histidine 195 in maintaining active-site asymmetry in presynaptic filaments of bacteriophage T4 UvsX recombinase.

    PubMed

    Farb, Joshua N; Morrical, Scott W

    2009-01-16

    Recombinases of the highly conserved RecA/Rad51 family play central roles in homologous recombination and DNA double-stranded break repair. RecA/Rad51 enzymes form presynaptic filaments on single-stranded DNA (ssDNA) that are allosterically activated to catalyze ATPase and DNA strand-exchange reactions. Information is conveyed between DNA- and ATP-binding sites, in part, by a highly conserved glutamine residue (Gln194 in Escherichia coli RecA) that acts as an allosteric switch. The T4 UvsX protein is a divergent RecA ortholog and contains histidine (His195) in place of glutamine at the allosteric switch position. UvsX and RecA catalyze similar strand-exchange reactions, but differ in other properties. UvsX produces both ADP and AMP as products of its ssDNA-dependent ATPase activity--a property that is unique among characterized recombinases. Details of the kinetics of ssDNA-dependent ATP hydrolysis reactions indicate that UvsX-ssDNA presynaptic filaments are asymmetric and contain two classes of ATPase active sites: one that generates ADP, and another that generates AMP. Active-site asymmetry is reduced by mutations at the His195 position, since UvsX-H195Q and UvsX-H195A mutants both exhibit stronger ssDNA-dependent ATPase activity, with lower cooperativity and markedly higher ADP/AMP product ratios, than wild-type UvsX. Reduced active-site asymmetry correlates strongly with reduced ssDNA-binding affinity and DNA strand-exchange activity in both H195Q and H195A mutants. These and other results support a model in which allosteric switch residue His195 controls the formation of an asymmetric conformation of UvsX-ssDNA filaments that is active in DNA strand exchange. The implications of our findings for UvsX recombination functions, and for RecA functions in general, are discussed.

  1. Decidable and undecidable arithmetic functions in actin filament networks

    NASA Astrophysics Data System (ADS)

    Schumann, Andrew

    2018-01-01

    The plasmodium of Physarum polycephalum is very sensitive to its environment, and reacts to stimuli with appropriate motions. Both the sensory and motor stages of these reactions are explained by hydrodynamic processes, based on fluid dynamics, with the participation of actin filament networks. This paper is devoted to actin filament networks as a computational medium. The point is that actin filaments, with contributions from many other proteins like myosin, are sensitive to extracellular stimuli (attractants as well as repellents), and appear and disappear at different places in the cell to change aspects of the cell structure—e.g. its shape. By assembling and disassembling actin filaments, some unicellular organisms, like Amoeba proteus, can move in response to various stimuli. As a result, these organisms can be considered a simple reversible logic gate—extracellular signals being its inputs and motions its outputs. In this way, we can implement various logic gates on amoeboid behaviours. These networks can embody arithmetic functions within p-adic valued logic. Furthermore, within these networks we can define the so-called diagonalization for deducing undecidable arithmetic functions.

  2. Scaling of F-actin network rheology to probe single filament elasticity and dynamics.

    PubMed

    Gardel, M L; Shin, J H; MacKintosh, F C; Mahadevan, L; Matsudaira, P A; Weitz, D A

    2004-10-29

    The linear and nonlinear viscoelastic response of networks of cross-linked and bundled cytoskeletal filaments demonstrates remarkable scaling with both frequency and applied prestress, which helps elucidate the origins of the viscoelasticity. The frequency dependence of the shear modulus reflects the underlying single-filament relaxation dynamics for 0.1-10 rad/sec. Moreover, the nonlinear strain stiffening of such networks exhibits a universal form as a function of prestress; this is quantitatively explained by the full force-extension relation of single semiflexible filaments.

  3. The Medicago truncatula GRAS protein RAD1 supports arbuscular mycorrhiza symbiosis and Phytophthora palmivora susceptibility.

    PubMed

    Rey, Thomas; Bonhomme, Maxime; Chatterjee, Abhishek; Gavrin, Aleksandr; Toulotte, Justine; Yang, Weibing; André, Olivier; Jacquet, Christophe; Schornack, Sebastian

    2017-12-16

    The roots of most land plants are colonized by symbiotic arbuscular mycorrhiza (AM) fungi. To facilitate this symbiosis, plant genomes encode a set of genes required for microbial perception and accommodation. However, the extent to which infection by filamentous root pathogens also relies on some of these genes remains an open question. Here, we used genome-wide association mapping to identify genes contributing to colonization of Medicago truncatula roots by the pathogenic oomycete Phytophthora palmivora. Single-nucleotide polymorphism (SNP) markers most significantly associated with plant colonization response were identified upstream of RAD1, which encodes a GRAS transcription regulator first negatively implicated in root nodule symbiosis and recently identified as a positive regulator of AM symbiosis. RAD1 transcript levels are up-regulated both in response to AM fungus and, to a lower extent, in infected tissues by P. palmivora where its expression is restricted to root cortex cells proximal to pathogen hyphae. Reverse genetics showed that reduction of RAD1 transcript levels as well as a rad1 mutant are impaired in their full colonization by AM fungi as well as by P. palmivora. Thus, the importance of RAD1 extends beyond symbiotic interactions, suggesting a general involvement in M. truncatula microbe-induced root development and interactions with unrelated beneficial and detrimental filamentous microbes. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  4. MinCD cell division proteins form alternating co-polymeric cytomotive filaments

    PubMed Central

    Ghosal, Debnath; Trambaiolo, Daniel; Amos, Linda A.; Löwe, Jan

    2014-01-01

    Summary During bacterial cell division, filaments of the tubulin-like protein FtsZ assemble at midcell to form the cytokinetic Z-ring. Its positioning is regulated by the oscillation of MinCDE proteins. MinC is activated by MinD through an unknown mechanism and prevents Z-ring assembly anywhere but midcell. Here, using X-ray crystallography, electron microscopy and in vivo analyses we show that MinD activates MinC by forming a new class of alternating copolymeric filaments that show similarity to eukaryotic septin filaments A non-polymerising mutation in MinD causes aberrant cell division in E. coli. MinCD copolymers bind to membrane, interact with FtsZ, and are disassembled by MinE. Imaging a functional msfGFP-MinC fusion protein in MinE deleted cells reveals filamentous structures. EM imaging of our reconstitution of the MinCD-FtsZ interaction on liposome surfaces reveals a plausible mechanism for regulation of FtsZ ring assembly by MinCD copolymers. PMID:25500731

  5. Characterization of gamma-tubulin filaments in mammalian cells.

    PubMed

    Lindström, Lisa; Alvarado-Kristensson, Maria

    2018-01-01

    Overexpression of γ-tubulin leads to the formation of filaments, but nothing is known about such filaments with regard to possible presence in cells, structure and probable dynamics. Here, we used mammalian cell lines to investigate the ability of γ-tubulin to form filaments. We found that γ-tubulin produces fibers called γ-tubules in a GTP-dependent manner and that γ-tubules are made up of pericentrin and the γ-tubulin complex proteins 2, 3, 5 and 6. Furthermore, we noted that the number of cells with cytosolic γ-tubules is increased in non-dividing cells. Our experiments showed that γ-tubules are polar structures that have a low regrowth rate compared to microtubules. Also, we observed that γ-tubules were disassembled by treatment with cold, colcemid, citral dimethyl acetal, dimethyl fumarate or mutation of γ-tubulin GTPase domain, but were increased in number by treatment with taxol or by stable expression of the γ-tubulin 1-333 GTPase domain. Our results demonstrate that γ-tubulin forms filaments, and such assembly is facilitated by the GTPase domain of γ-tubulin. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  6. Ubiquitylation by Trim32 causes coupled loss of desmin, Z-bands, and thin filaments in muscle atrophy

    PubMed Central

    Cohen, Shenhav; Zhai, Bo; Gygi, Steven P.

    2012-01-01

    During muscle atrophy, myofibrillar proteins are degraded in an ordered process in which MuRF1 catalyzes ubiquitylation of thick filament components (Cohen et al. 2009. J. Cell Biol. http://dx.doi.org/10.1083/jcb.200901052). Here, we show that another ubiquitin ligase, Trim32, ubiquitylates thin filament (actin, tropomyosin, troponins) and Z-band (α-actinin) components and promotes their degradation. Down-regulation of Trim32 during fasting reduced fiber atrophy and the rapid loss of thin filaments. Desmin filaments were proposed to maintain the integrity of thin filaments. Accordingly, we find that the rapid destruction of thin filament proteins upon fasting was accompanied by increased phosphorylation of desmin filaments, which promoted desmin ubiquitylation by Trim32 and degradation. Reducing Trim32 levels prevented the loss of both desmin and thin filament proteins. Furthermore, overexpression of an inhibitor of desmin polymerization induced disassembly of desmin filaments and destruction of thin filament components. Thus, during fasting, desmin phosphorylation increases and enhances Trim32-mediated degradation of the desmin cytoskeleton, which appears to facilitate the breakdown of Z-bands and thin filaments. PMID:22908310

  7. Modeling operational behavior of a disassembly line

    NASA Astrophysics Data System (ADS)

    Kizilkaya, Elif A.; Gupta, Surendra M.

    2004-12-01

    In this paper we present a dynamic kanban (pull) system specifically developed for disassembly lines. This type of kanban system is much more complex than the traditional kanban system used in assembly lines. For instance, unlike the assembly line where the external demand occurs only at the last station, the demands in the disassembly case also occur at any of the intermittent stations. The reason is that as a product moves on the disassembly line, various parts are disassembled at every station and accumulated at that station. Therefore, there are as many demand sources as there are number of parts. We consider a case example involving the end-of-life products. Based on the precedence relationships and other criteria such as hazardous properties of the parts, we balance the disassembly line. The results of the disassembly line-balancing problem (DLBP) are used as input to the proposed dynamic kanban system for disassembly line (DKSDL). We compare the performance of the DKSDL to the modified kanban system for disassembly line (MKSDL), which was previously introduced by the authors. We show, via simulation, that the DKSDL is far superior to MKSDL considered.

  8. CRISPR Technology Reveals RAD(51)-ical Mechanisms of Repair in Roundworms: An Educational Primer for Use with "Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans".

    PubMed

    Turcotte, Carolyn A; Andrews, Nicolas P; Sloat, Solomon A; Checchi, Paula M

    2016-11-01

    The mechanisms cells use to maintain genetic fidelity via DNA repair and the accuracy of these processes have garnered interest from scientists engaged in basic research to clinicians seeking improved treatment for cancer patients. Despite the continued advances, many details of DNA repair are still incompletely understood. In addition, the inherent complexity of DNA repair processes, even at the most fundamental level, makes it a challenging topic. This primer is meant to assist both educators and students in using a recent paper, "Promotion of homologous recombination by SWS-1 in complex with RAD-51 paralogs in Caenorhabditis elegans," to understand mechanisms of DNA repair. The goals of this primer are to highlight and clarify several key techniques utilized, with special emphasis on the clustered, regularly interspaced, short palindromic repeats technique and the ways in which it has revolutionized genetics research, as well as to provide questions for deeper in-class discussion. Copyright © 2016 by the Genetics Society of America.

  9. Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin*

    PubMed Central

    Courson, David S.; Rock, Ronald S.

    2010-01-01

    Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and α-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. α-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo. PMID:20551315

  10. Generation of control sequences for a pilot-disassembly system

    NASA Astrophysics Data System (ADS)

    Seliger, Guenther; Kim, Hyung-Ju; Keil, Thomas

    2002-02-01

    Closing the product and material cycles has emerged as a paradigm for industry in the 21st century. Disassembly plays a key role in a life cycle economy since it enables the recovery of resources. A partly automated disassembly system should adapt to a large variety of products and different degrees of devaluation. Also the amounts of products to be disassembled can vary strongly. To cope with these demands an approach to generate on-line disassembly control sequences will be presented. In order to react on these demands the technological feasibility is considered within a procedure for the generation of disassembly control sequences. Procedures are designed to find available and technologically feasible disassembly processes. The control system is formed by modularised and parameterised control units in the cell level within the entire control architecture. In the first development stage product and process analyses at the sample product washing machine were executed. Furthermore a generalized disassembly process was defined. Afterwards these processes were structured in primary and secondary functions. In the second stage the disassembly control at the technological level was investigated. Factors were the availability of the disassembly tools and the technological feasibility of the disassembly processes within the disassembly system. Technical alternative disassembly processes are determined as a result of availability of the tools and technological feasibility of processes. The fourth phase was the concept for the generation of the disassembly control sequences. The approach will be proved in a prototypical disassembly system.

  11. Mutation-specific effects on thin filament length in thin filament myopathy.

    PubMed

    Winter, Josine M de; Joureau, Barbara; Lee, Eun-Jeong; Kiss, Balázs; Yuen, Michaela; Gupta, Vandana A; Pappas, Christopher T; Gregorio, Carol C; Stienen, Ger J M; Edvardson, Simon; Wallgren-Pettersson, Carina; Lehtokari, Vilma-Lotta; Pelin, Katarina; Malfatti, Edoardo; Romero, Norma B; Engelen, Baziel G van; Voermans, Nicol C; Donkervoort, Sandra; Bönnemann, C G; Clarke, Nigel F; Beggs, Alan H; Granzier, Henk; Ottenheijm, Coen A C

    2016-06-01

    Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation. We investigated the sarcomere length-dependence of force, a functional assay that provides insights into the contractile strength of muscle fibers as well as the length of the thin filaments, in muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. Lower force generation was observed in muscle fibers from patients of all genotypes. In a subset of patients who harbor mutations in NEB and ACTA1, the lower force was associated with downward shifted force-sarcomere length relations, indicative of shorter thin filaments. Confocal microscopy confirmed shorter thin filaments in muscle fibers of these patients. A conditional Neb knockout mouse model, which recapitulates thin filament myopathy, revealed a compensatory mechanism; the lower force generation that was associated with shorter thin filaments was compensated for by increasing the number of sarcomeres in series. This allowed muscle fibers to operate at a shorter sarcomere length and maintain optimal thin-thick filament overlap. These findings might provide a novel direction for the development of therapeutic strategies for thin filament myopathy patients with shortened thin filament lengths. Ann Neurol 2016;79:959-969. © 2016 American Neurological Association.

  12. Mutation-Specific Effects on Thin Filament Length in Thin Filament Myopathy

    PubMed Central

    de Winter, Josine M.; Joureau, Barbara; Lee, Eun-Jeong; Kiss, Balázs; Yuen, Michaela; Gupta, Vandana A.; Pappas, Christopher T.; Gregorio, Carol C.; Stienen, Ger J. M.; Edvardson, Simon; Wallgren-Pettersson, Carina; Lehtokari, Vilma-Lotta; Pelin, Katarina; Malfatti, Edoardo; Romero, Norma B.; van Engelen, Baziel G.; Voermans, Nicol C.; Donkervoort, Sandra; Bönnemann, C. G.; Clarke, Nigel F.; Beggs, Alan H.; Granzier, Henk; Ottenheijm, Coen A. C.

    2016-01-01

    Objective Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation. Methods We investigated the sarcomere length-dependence of force, a functional assay that provides insights into the contractile strength of muscle fibers as well as the length of the thin filaments, in muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. Results Lower force generation was observed in muscle fibers from patients of all genotypes. In a subset of patients who harbor mutations in NEB and ACTA1, the lower force was associated with downward shifted force–sarcomere length relations, indicative of shorter thin filaments. Confocal microscopy confirmed shorter thin filaments in muscle fibers of these patients. A conditional Neb knockout mouse model, which recapitulates thin filament myopathy, revealed a compensatory mechanism; the lower force generation that was associated with shorter thin filaments was compensated for by increasing the number of sarcomeres in series. This allowed muscle fibers to operate at a shorter sarcomere length and maintain optimal thin–thick filament overlap. Interpretation These findings might provide a novel direction for the development of therapeutic strategies for thin filament myopathy patients with shortened thin filament lengths. PMID:27074222

  13. Cellular level robotic surgery: Nanodissection of intermediate filaments in live keratinocytes.

    PubMed

    Yang, Ruiguo; Song, Bo; Sun, Zhiyong; Lai, King Wai Chiu; Fung, Carmen Kar Man; Patterson, Kevin C; Seiffert-Sinha, Kristina; Sinha, Animesh A; Xi, Ning

    2015-01-01

    We present the nanosurgery on the cytoskeleton of live cells using AFM based nanorobotics to achieve adhesiolysis and mimic the effect of pathophysiological modulation of intercellular adhesion. Nanosurgery successfully severs the intermediate filament bundles and disrupts cell-cell adhesion similar to the desmosomal protein disassembly in autoimmune disease, or the cationic modulation of desmosome formation. Our nanomechanical analysis revealed that adhesion loss results in a decrease in cellular stiffness in both cases of biochemical modulation of the desmosome junctions and mechanical disruption of intercellular adhesion, supporting the notion that intercellular adhesion through intermediate filaments anchors the cell structure as focal adhesion does and that intermediate filaments are integral components in cell mechanical integrity. The surgical process could potentially help reveal the mechanism of autoimmune pathology-induced cell-cell adhesion loss as well as its related pathways that lead to cell apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. The GTP binding proteins Gem and Rad are negative regulators of the Rho–Rho kinase pathway

    PubMed Central

    Ward, Yvona; Yap, Seow-Fong; Ravichandran, V.; Matsumura, Fumio; Ito, Masaaki; Spinelli, Beth; Kelly, Kathleen

    2002-01-01

    The cytoskeletal changes that alter cellular morphogenesis and motility depend upon a complex interplay among molecules that regulate actin, myosin, and other cytoskeletal components. The Rho family of GTP binding proteins are important upstream mediators of cytoskeletal organization. Gem and Rad are members of another family of small GTP binding proteins (the Rad, Gem, and Kir family) for which biochemical functions have been mostly unknown. Here we show that Gem and Rad interface with the Rho pathway through association with the Rho effectors, Rho kinase (ROK) α and β. Gem binds ROKβ independently of RhoA in the ROKβ coiled-coil region adjacent to the Rho binding domain. Expression of Gem inhibited ROKβ-mediated phosphorylation of myosin light chain and myosin phosphatase, but not LIM kinase, suggesting that Gem acts by modifying the substrate specificity of ROKβ. Gem or Rad expression led to cell flattening and neurite extension in N1E-115 neuroblastoma cells. In interference assays, Gem opposed ROKβ- and Rad opposed ROKα-mediated cell rounding and neurite retraction. Gem did not oppose cell rounding initiated by ROKβ containing a deletion of the Gem binding region, demonstrating that Gem binding to ROKβ is required for the effects observed. In epithelial or fibroblastic cells, Gem or Rad expression resulted in stress fiber and focal adhesion disassembly. In addition, Gem reverted the anchorage-independent growth and invasiveness of Dbl-transformed fibroblasts. These results identify physiological roles for Gem and Rad in cytoskeletal regulation mediated by ROK. PMID:11956230

  15. Research on automated disassembly technology for waste LCD

    NASA Astrophysics Data System (ADS)

    Qin, Qin; Zhu, Dongdong; Wang, Jingwei; Dou, Jianfang; Wang, Sujuan; Tu, Zimei

    2017-11-01

    In the field of Waste LCD disassembling and recycling, there are existing two major problems: 1) disassembling waste LCD mainly depends on manually mechanical crushing; 2) the resource level is not high. In order to deal with the above problems, in this paper, we develop an efficient, safe and automated waste LCD disassembling assembly line technology. This technology can disassembly and classify mainstream LCD into four components, which are liquid crystal display panels, housings and metal shield, PCB assembly. It can also disassembly many kinds of waste LCD. Compared with the traditional cooperation of manual labor and electric tools method, our proposed technology can significantly improve disassembling efficiency and demonstrate good prospects and promotional value.

  16. Impact of different disassembly line balancing algorithms on the performance of dynamic kanban system for disassembly line

    NASA Astrophysics Data System (ADS)

    Kizilkaya, Elif A.; Gupta, Surendra M.

    2005-11-01

    In this paper, we compare the impact of different disassembly line balancing (DLB) algorithms on the performance of our recently introduced Dynamic Kanban System for Disassembly Line (DKSDL) to accommodate the vagaries of uncertainties associated with disassembly and remanufacturing processing. We consider a case study to illustrate the impact of various DLB algorithms on the DKSDL. The approach to the solution, scenario settings, results and the discussions of the results are included.

  17. Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae.

    PubMed

    Stein, Alexis; Kalifa, Lidza; Sia, Elaine A

    2015-11-01

    Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs.

  18. Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae

    PubMed Central

    Stein, Alexis; Kalifa, Lidza; Sia, Elaine A.

    2015-01-01

    Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs. PMID:26540255

  19. Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane

    PubMed Central

    Gokhin, David S.; Nowak, Roberta B.; Khoory, Joseph A.; de la Piedra, Alfonso; Ghiran, Ionita C.; Fowler, Velia M.

    2015-01-01

    Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (∼25–30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ∼60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane. PMID:25717184

  20. Multikanban model for disassembly line with demand fluctuation

    NASA Astrophysics Data System (ADS)

    Udomsawat, Gun; Gupta, Surendra M.; Al-Turki, Yousef A. Y.

    2004-02-01

    In recent years, the continuous growth in consumer waste and dwindling natural resources has seriously threatened the environment. Realizing this, several countries have passed regulations that force manufacturers not only to manufacture environmentally conscious products, but also to take back their used products from consumers so that the components and materials recovered from the products may be reused and/or recycled. Disassembly plays an important role in product recovery. A disassembly line is perhaps the most suitable setting for disassembly of products in large quantities. Because a disassembly line has a tendency to generate excessive inventory, employing a kanban system can reduce the inventory level and let the system run more efficiently. A disassembly line is quite different from an assembly line. For example, not only can the demand arrive at the last station, it can also arrive at any of the other stations in the system. The demand for a component on the disassembly line could fluctuate widely. In fact, there are many other complicating matters that need to be considered to implement the concept of kanbans in such an environment. In this paper, we discuss the complications that are unique to a disassembly line. We discuss the complications in utilizing the conventional production control mechanisms in a disassembly line setting. We then show how to overcome them by implementing kanbans in a disassembly line setting with demand fluctuation and introduce the concept of multi-kanban mechanism. We demonstrate its effectiveness using a simulation model. An example is presented to illustrate the concept.

  1. Computational algorithm to evaluate product disassembly cost index

    NASA Astrophysics Data System (ADS)

    Zeid, Ibrahim; Gupta, Surendra M.

    2002-02-01

    Environmentally conscious manufacturing is an important paradigm in today's engineering practice. Disassembly is a crucial factor in implementing this paradigm. Disassembly allows the reuse and recycling of parts and products that reach their death after their life cycle ends. There are many questions that must be answered before a disassembly decision can be reached. The most important question is economical. The cost of disassembly versus the cost of scrapping a product is always considered. This paper develops a computational tool that allows decision-makers to calculate the disassembly cost of a product. The tool makes it simple to perform 'what if' scenarios fairly quickly. The tool is Web based and has two main parts. The front-end part is a Web page and runs on the client side in a Web browser, while the back-end part is a disassembly engine (servlet) that has disassembly knowledge and costing algorithms and runs on the server side. The tool is based on the client/server model that is pervasively utilized throughout the World Wide Web. An example is used to demonstrate the implementation and capabilities of the tool.

  2. The influence of Filaments in the Private Flux Region on Divertor Power and Particle Deposition

    NASA Astrophysics Data System (ADS)

    Harrison, James

    2014-10-01

    Recent advances in imaging of the MAST divertor have revealed, for the first time, evidence for filaments in the private flux region (PFR). Detailed analysis of the image data shows 3 distinct types of fluctuations occurring within the divertor volume: highly sheared filaments in the SOL originating from the outer midplane, high frequency (>50 kHz) filaments near the separatrix of the outer divertor leg and filaments in the private flux region originating from inner divertor leg. With the need to extrapolate divertor performance from existing machines to future devices, these observations can contribute to our quantitative understanding of transport in the PFR. In particular, they suggest that transport in the PFR is, at least in part, driven by turbulence, which may not be well captured by the Eich/Wagner description of the divertor footprint, expressed in terms of exponential decay in space above the X-point and Gaussian spreading below the X-point. The PFR filaments are observed to move largely parallel with the flux surfaces in a way equivalent to a toroidal angular velocity of order 2 ×104 rad/s in H-mode, and slower by a factor of order 2 in L-mode. During their transit parallel to the flux surfaces across the PFR, the filaments eject plasma in bursts, away from the separatrix, deeper into the private flux region. Correlation analysis suggests that they are generated by processes local to the inner divertor leg, as there is a weak correlation between fluctuations in the SOL and PFR above what is expected from line integration effects. Scaling of filament properties with machine operating parameters, such as plasma current, density and auxiliary heating power will be presented, together with a comparison with data from divertor Langmuir probes and IR thermography to estimate the role PFR filaments play in determining the width of the divertor footprint.

  3. Damage-Free Relief-Valve Disassembly

    NASA Technical Reports Server (NTRS)

    Haselmaier, H.

    1986-01-01

    Tool safely disassembles relief valves without damage to sensitive parts. Relief-valve disassembly tool used to extract valve nozzle from its housing. Holding device on tool grops nozzle. When user strikes hammer against impact disk, holding device pulls nozzle from press fit. Previously, nozzle dislodged by striking spindle above it, but practice often damaged retaining screw. New tool removes nozzle directly. With minor modifications, tool adapted to valves from different manufacturers.

  4. Recycling of electrical motors by automatic disassembly

    NASA Astrophysics Data System (ADS)

    Karlsson, Björn; Järrhed, Jan-Ove

    2000-04-01

    This paper presents a robotized workstation for end-of-life treatment of electrical motors with an electrical effect of about 1 kW. These motors can, for example, be found in washing machines and in industry. There are two main steps in the work. The first step is an inspection whereby the functionality of the motor is checked and classification either for re-use or for disassembly is done. In the second step the motors classified for disassembly are disassembled in a robotized automatic station. In the initial step measurements are performed during a start-up sequence of about 1 s. By measuring the rotation speed and the current and voltage of the three phases of the motor classification for either reuse or disassembly can be done. During the disassembly work, vision data are fused in order to classify the motors according to their type. The vision system also feeds the control system of the robot with various object co-ordinates, to facilitate correct operation of the robot. Finally, tests with a vision system and eddy-current equipment are performed to decide whether all copper has been removed from the stator.

  5. Mechanical force antagonizes the inhibitory effects of RecX on RecA filament formation in Mycobacterium tuberculosis

    PubMed Central

    Le, Shimin; Chen, Hu; Zhang, Xinghua; Chen, Jin; Patil, K. Neelakanteshwar; Muniyappa, Kalappa; Yan, Jie

    2014-01-01

    Efficient bacterial recombinational DNA repair involves rapid cycles of RecA filament assembly and disassembly. The RecX protein plays a crucial inhibitory role in RecA filament formation and stability. As the broken ends of DNA are tethered during homologous search, RecA filaments assembled at the ends are likely subject to force. In this work, we investigated the interplay between RecX and force on RecA filament formation and stability. Using magnetic tweezers, at single molecular level, we found that Mycobacterium tuberculosis (Mt) RecX could catalyze stepwise de-polymerization of preformed MtRecA filament in the presence of ATP hydrolysis at low forces (<7 pN). However, applying larger forces antagonized the inhibitory effects of MtRecX, and a partially de-polymerized MtRecA filament could re-polymerize in the presence of MtRecX, which cannot be explained by previous models. Theoretical analysis of force-dependent conformational free energies of naked ssDNA and RecA nucleoprotein filament suggests that mechanical force stabilizes RecA filament, which provides a possible mechanism for the observation. As the antagonizing effect of force on the inhibitory function of RecX takes place in a physiological range; these findings broadly suggest a potential mechanosensitive regulation during homologous recombination. PMID:25294832

  6. Electronic waste disassembly with industrial waste heat.

    PubMed

    Chen, Mengjun; Wang, Jianbo; Chen, Haiyian; Ogunseitan, Oladele A; Zhang, Mingxin; Zang, Hongbin; Hu, Jiukun

    2013-01-01

    Waste printed circuit boards (WPCBs) are resource-rich but hazardous, demanding innovative strategies for post-consumer collection, recycling, and mining for economically precious constituents. A novel technology for disassembling electronic components from WPCBs is proposed, using hot air to melt solders and to separate the components and base boards. An automatic heated-air disassembling equipment was designed to operate at a heating source temperature at a maximum of 260 °C and an inlet pressure of 0.5 MPa. A total of 13 individual WPCBs were subjected to disassembling tests at different preheat temperatures in increments of 20 °C between 80 and 160 °C, heating source temperatures ranging from 220 to 300 °C in increments of 20 °C, and incubation periods of 1, 2, 4, 6, or 8 min. For each experimental treatment, the disassembly efficiency was calculated as the ratio of electronic components released from the board to the total number of its original components. The optimal preheat temperature, heating source temperature, and incubation period to disassemble intact components were 120 °C, 260 °C, and 2 min, respectively. The disassembly rate of small surface mount components (side length ≤ 3 mm) was 40-50% lower than that of other surface mount components and pin through hole components. On the basis of these results, a reproducible and sustainable industrial ecological protocol using steam produced by industrial exhaust heat coupled to electronic-waste recycling is proposed, providing an efficient, promising, and green method for both electronic component recovery and industrial exhaust heat reutilization.

  7. Protein Phosphatase Pph3 and Its Regulatory Subunit Psy2 Regulate Rad53 Dephosphorylation and Cell Morphogenesis during Recovery from DNA Damage in Candida albicans ▿

    PubMed Central

    Sun, Ling Ling; Li, Wan Jie; Wang, Hai Tao; Chen, Jie; Deng, Ping; Wang, Yue; Sang, Jian Li

    2011-01-01

    The ability of the pathogenic fungus Candida albicans to switch cellular morphologies is important for infection and virulence. Recent studies have revealed that C. albicans yeast cells can switch to filamentous growth under genotoxic stress in a manner dependent on the DNA replication/damage checkpoint. Here, we have investigated the functions of Pph3 (orf19.4378) and Psy2 (orf19.3685), whose orthologues in Saccharomyces cerevisiae mediate the dephosphorylation of the DNA damage checkpoint kinase Rad53 and the histone variant H2AX during recovery from DNA damage. Deleting PPH3 or PSY2 causes hypersensitivity to DNA-damaging agents, including cisplatin, methylmethane sulfonate (MMS), and UV light. In addition, pph3Δ and psy2Δ cells exhibit strong filamentous growth under genotoxic stress. Flow cytometry analysis shows that the mutant cells have lost the ability to adapt to genotoxic stress and remain arrested even after the stress is withdrawn. Furthermore, we show that Pph3 and Psy2 are required for the dephosphorylation of Rad53, but not H2AX, during DNA damage recovery. Taken together, these results show that C. albicans Pph3 and Psy2 have important roles in mediating genotoxin-induced filamentous growth and regulating Rad53 dephosphorylation. PMID:21890819

  8. Multi-kanban mechanism for appliance disassembly

    NASA Astrophysics Data System (ADS)

    Udomsawat, Gun; Gupta, Surendra M.

    2005-11-01

    The use of household appliances continues to rise every year. A significant number of End-Of-Life (EOL) appliances are generated because of the introduction of newer models that are more attractive, efficient and affordable. Others are, of course, generated when they become non-functional. Many regulations encourage recycling of EOL appliances to reduce the amount of waste sent to landfills. In addition, EOL appliances offer the appliance manufacturing and remanufacturing industries a source of less expensive raw materials and components. For this reason product recovery has become a subject of interest during the past decade. In this paper, we study the disassembly line for appliance disassembly. We discuss and incorporate some of the complications that are inherent in disassembly line including product arrival, demand arrival, inventory fluctuation and production control mechanisms. We show how to overcome such complications by implementing a multi-kanban system in the appliance disassembly line setting. The multi-kanban system (MKS) relies on dynamic routing of kanbans according to the state of the system. We investigate the multi-kanban mechanism using simulation and explore the effect of product mix on performance of the traditional push system (TPS) and MKS in terms of controlling the system's inventory while attempting to achieve a decent customer service level.

  9. An Overview of the Molecular Mechanisms of Recombinational DNA Repair

    PubMed Central

    Kowalczykowski, Stephen C.

    2015-01-01

    Recombinational DNA repair is a universal aspect of DNA metabolism and is essential for genomic integrity. It is a template-directed process that uses a second chromosomal copy (sister, daughter, or homolog) to ensure proper repair of broken chromosomes. The key steps of recombination are conserved from phage through human, and an overview of those steps is provided in this review. The first step is resection by helicases and nucleases to produce single-stranded DNA (ssDNA) that defines the homologous locus. The ssDNA is a scaffold for assembly of the RecA/RAD51 filament, which promotes the homology search. On finding homology, the nucleoprotein filament catalyzes exchange of DNA strands to form a joint molecule. Recombination is controlled by regulating the fate of both RecA/RAD51 filaments and DNA pairing intermediates. Finally, intermediates that mature into Holliday structures are disjoined by either nucleolytic resolution or topological dissolution. PMID:26525148

  10. Hypoxia transiently sequesters mps1 and polo to collagenase-sensitive filaments in Drosophila prometaphase oocytes.

    PubMed

    Gilliland, William D; Vietti, Dana L; Schweppe, Nicole M; Guo, Fengli; Johnson, Teri J; Hawley, R Scott

    2009-10-22

    The protein kinases Mps1 and Polo, which are required for proper cell cycle regulation in meiosis and mitosis, localize to numerous ooplasmic filaments during prometaphase in Drosophila oocytes. These filaments first appear throughout the oocyte at the end of prophase and are disassembled after egg activation. We showed here that Mps1 and Polo proteins undergo dynamic and reversible localization to static ooplasmic filaments as part of an oocyte-specific response to hypoxia. The observation that Mps1- and Polo-associated filaments reappear in the same locations through multiple cycles of oxygen deprivation demonstrates that underlying structural components of the filaments must still be present during normoxic conditions. Using immuno-electron microscopy, we observed triple-helical binding of Mps1 to numerous electron-dense filaments, with the gold label wrapped around the outside of the filaments like a garland. In addition, we showed that in live oocytes the relocalization of Mps1 and Polo to filaments is sensitive to injection of collagenase, suggesting that the structural components of the filaments are composed of collagen-like fibrils. However, the collagen-like genes we have been able to test so far (vkg and CG42453) did not appear to be associated with the filaments, demonstrating that the collagenase-sensitive component of the filaments is one of a number of other Drosophila proteins bearing a collagenase cleavage site. Finally, as hypoxia is known to cause Mps1 protein to accumulate at kinetochores in syncytial embryos, we also show that GFP-Polo accumulates at both kinetochores and centrosomes in hypoxic syncytial embryos. These findings identify both a novel cellular structure (the ooplasmic filaments) as well as a new localization pattern for Mps1 and Polo and demonstrate that hypoxia affects Polo localization in Drosophila.

  11. An Intelligent Agent-Controlled and Robot-Based Disassembly Assistant

    NASA Astrophysics Data System (ADS)

    Jungbluth, Jan; Gerke, Wolfgang; Plapper, Peter

    2017-09-01

    One key for successful and fluent human-robot-collaboration in disassembly processes is equipping the robot system with higher autonomy and intelligence. In this paper, we present an informed software agent that controls the robot behavior to form an intelligent robot assistant for disassembly purposes. While the disassembly process first depends on the product structure, we inform the agent using a generic approach through product models. The product model is then transformed to a directed graph and used to build, share and define a coarse disassembly plan. To refine the workflow, we formulate “the problem of loosening a connection and the distribution of the work” as a search problem. The created detailed plan consists of a sequence of actions that are used to call, parametrize and execute robot programs for the fulfillment of the assistance. The aim of this research is to equip robot systems with knowledge and skills to allow them to be autonomous in the performance of their assistance to finally improve the ergonomics of disassembly workstations.

  12. Understanding nucleotide-regulated FtsZ filament dynamics and the monomer assembly switch with large-scale atomistic simulations.

    PubMed

    Ramírez-Aportela, Erney; López-Blanco, José Ramón; Andreu, José Manuel; Chacón, Pablo

    2014-11-04

    Bacterial cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for cell division. Here, we study their dynamics using unbiased atomistic molecular simulations from representative filament crystal structures. In agreement with experimental data, we find different filament curvatures that are supported by a nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby burying the nucleotide, the differently curved GDP-FtsZ filaments exhibit a heterogeneous distribution of open and closed interfaces between monomers. We identify a coordinated Mg(2+) ion as the key structural element in closing the nucleotide site and stabilizing GTP filaments, whereas the loss of the contacts with loop T7 from the next monomer in GDP filaments leads to open interfaces that are more prone to depolymerization. We monitored the FtsZ monomer assembly switch, which involves opening/closing of the cleft between the C-terminal domain and the H7 helix, and observed the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive conformation. This result validates the proposed switch between the low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ conformation within filaments. Finally, we observed how the antibiotic PC190723 suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces, thus stabilizing the filament. Our studies provide detailed structural and dynamic insights into modulation of both the intrinsic curvature of the FtsZ filaments and the molecular switch coupled to the high-affinity end-wise association of FtsZ monomers.

  13. Profilin-Dependent Nucleation and Assembly of Actin Filaments Controls Cell Elongation in Arabidopsis1[OPEN

    PubMed Central

    Cao, Lingyan; Blanchoin, Laurent; Staiger, Christopher J.

    2016-01-01

    Actin filaments in plant cells are incredibly dynamic; they undergo incessant remodeling and assembly or disassembly within seconds. These dynamic events are choreographed by a plethora of actin-binding proteins, but the exact mechanisms are poorly understood. Here, we dissect the contribution of Arabidopsis (Arabidopsis thaliana) PROFILIN1 (PRF1), a conserved actin monomer-binding protein, to actin organization and single filament dynamics during axial cell expansion of living epidermal cells. We found that reduced PRF1 levels enhanced cell and organ growth. Surprisingly, we observed that the overall frequency of nucleation events in prf1 mutants was dramatically decreased and that a subpopulation of actin filaments that assemble at high rates was reduced. To test whether profilin cooperates with plant formin proteins to execute actin nucleation and rapid filament elongation in cells, we used a pharmacological approach. Here, we used Small Molecule Inhibitor of Formin FH2 (SMIFH2), after validating its mode of action on a plant formin in vitro, and observed a reduced nucleation frequency of actin filaments in live cells. Treatment of wild-type epidermal cells with SMIFH2 mimicked the phenotype of prf1 mutants, and the nucleation frequency in prf1-2 mutant was completely insensitive to these treatments. Our data provide compelling evidence that PRF1 coordinates the stochastic dynamic properties of actin filaments by modulating formin-mediated actin nucleation and assembly during plant cell expansion. PMID:26574597

  14. Rad59-Facilitated Acquisition of Y′ Elements by Short Telomeres Delays the Onset of Senescence

    PubMed Central

    Churikov, Dmitri; Charifi, Ferose; Simon, Marie-Noëlle; Géli, Vincent

    2014-01-01

    Telomerase-negative yeasts survive via one of the two Rad52-dependent recombination pathways, which have distinct genetic requirements. Although the telomere pattern of type I and type II survivors is well characterized, the mechanistic details of short telomere rearrangement into highly evolved pattern observed in survivors are still missing. Here, we analyze immediate events taking place at the abruptly shortened VII-L and native telomeres. We show that short telomeres engage in pairing with internal Rap1-bound TG1–3-like tracts present between subtelomeric X and Y′ elements, which is followed by BIR-mediated non-reciprocal translocation of Y′ element and terminal TG1–3 repeats from the donor end onto the shortened telomere. We found that choice of the Y′ donor was not random, since both engineered telomere VII-L and native VI-R acquired Y′ elements from partially overlapping sets of specific chromosome ends. Although short telomere repair was associated with transient delay in cell divisions, Y′ translocation on native telomeres did not require Mec1-dependent checkpoint. Furthermore, the homeologous pairing between the terminal TG1–3 repeats at VII-L and internal repeats on other chromosome ends was largely independent of Rad51, but instead it was facilitated by Rad59 that stimulates Rad52 strand annealing activity. Therefore, Y′ translocation events taking place during presenescence are genetically separable from Rad51-dependent Y′ amplification process that occurs later during type I survivor formation. We show that Rad59-facilitated Y′ translocations on X-only telomeres delay the onset of senescence while preparing ground for type I survivor formation. PMID:25375789

  15. Common Breast Cancer Susceptibility Variants in LSP1 and RAD51L1 Are Associated with Mammographic Density Measures that Predict Breast Cancer Risk

    PubMed Central

    Vachon, Celine M.; Scott, Christopher G.; Fasching, Peter A.; Hall, Per; Tamimi, Rulla M.; Li, Jingmei; Stone, Jennifer; Apicella, Carmel; Odefrey, Fabrice; Gierach, Gretchen L.; Jud, Sebastian M.; Heusinger, Katharina; Beckmann, Matthias W.; Pollan, Marina; Fernández-Navarro, Pablo; González-Neira, Anna; Benítez, Javier; van Gils, Carla H.; Lokate, Mariëtte; Onland-Moret, N. Charlotte; Peeters, Petra H.M.; Brown, Judith; Leyland, Jean; Varghese, Jajini S.; Easton, Douglas F.; Thompson, Deborah J.; Luben, Robert N.; Warren, Ruth ML; Wareham, Nicholas J.; Loos, Ruth JF; Khaw, Kay-Tee; Ursin, Giske; Lee, Eunjung; Gayther, Simon A.; Ramus, Susan J.; Eeles, Rosalind A.; Leach, Martin O.; Kwan-Lim, Gek; Couch, Fergus J.; Giles, Graham G.; Baglietto, Laura; Krishnan, Kavitha; Southey, Melissa C.; Le Marchand, Loic; Kolonel, Laurence N.; Woolcott, Christy; Maskarinec, Gertraud; Haiman, Christopher A; Walker, Kate; Johnson, Nichola; McCormack, Valerie A.; Biong, Margarethe; Alnæs, Grethe I.G.; Gram, Inger Torhild; Kristensen, Vessela N.; Børresen-Dale, Anne-Lise; Lindström, Sara; Hankinson, Susan E.; Hunter, David J.; Andrulis, Irene L.; Knight, Julia A.; Boyd, Norman F.; Figueroa, Jonine D.; Lissowska, Jolanta; Wesolowska, Ewa; Peplonska, Beata; Bukowska, Agnieszka; Reszka, Edyta; Liu, JianJun; Eriksson, Louise; Czene, Kamila; Audley, Tina; Wu, Anna H.; Pankratz, V. Shane; Hopper, John L.; dos-Santos-Silva, Isabel

    2013-01-01

    Background Mammographic density adjusted for age and body mass index (BMI) is a heritable marker of breast cancer susceptibility. Little is known about the biological mechanisms underlying the association between mammographic density and breast cancer risk. We examined whether common low-penetrance breast cancer susceptibility variants contribute to inter-individual differences in mammographic density measures. Methods We established an international consortium (DENSNP) of 19 studies from 10 countries, comprising 16,895 Caucasian women, to conduct a pooled cross-sectional analysis of common breast cancer susceptibility variants in 14 independent loci and mammographic density measures. Dense and non-dense areas, and percent density, were measured using interactive-thresholding techniques. Mixed linear models were used to assess the association between genetic variants and the square roots of mammographic density measures adjusted for study, age, case status, body mass index (BMI) and menopausal status. Results Consistent with their breast cancer associations, the C-allele of rs3817198 in LSP1 was positively associated with both adjusted dense area (p=0.00005) and adjusted percent density (p=0.001) whereas the A-allele of rs10483813 in RAD51L1 was inversely associated with adjusted percent density (p=0.003), but not with adjusted dense area (p=0.07). Conclusion We identified two common breast cancer susceptibility variants associated with mammographic measures of radio-dense tissue in the breast gland. Impact We examined the association of 14 established breast cancer susceptibility loci with mammographic density phenotypes within a large genetic consortium and identified two breast cancer susceptibility variants, LSP1-rs3817198 and RAD51L1-rs10483813, associated with mammographic measures and in the same direction as the breast cancer association. PMID:22454379

  16. 19 CFR 181.132 - Disassembly.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... (CONTINUED) NORTH AMERICAN FREE TRADE AGREEMENT Rules of Origin § 181.132 Disassembly. (a) Treated as production. For purposes of implementing the rules of origin provisions of General Note 12, HTSUS, and...

  17. Risk of Contamination in Assembled vs Disassembled Instruments in Hip Arthroplasty Surgery

    PubMed Central

    Mayer, Ryan R.; Bederman, S. Samuel; Colin, Vincent M.; Berger, Martina M.; Cesario, Thomas C.; Schwarzkopf, Ran

    2018-01-01

    Background Periprosthetic joint infection (PJI) is one of the most common causes of revision total hip arthroplasty (THA) and associated with higher costs, prolonged pain, and worse clinical outcomes. Many factors have been linked to increased infection rates, one being the operative equipment and instrumentation used during the surgical procedure. With few arthroplasty instruments designed for complete disassembly and increasingly complex instrument designs, this study seeks to understand the effect that instrument disassembly plays on infection using disassembled and assembled standard femoral broach handles (BHs). Methods Two BHs, not designed for disassembly, were modified and then contaminated in the disassembled state with Geobacillus stearothermophilus vegetative-form bacteria and spores. Using both flash and standard sterilization cycles, the BHs were steam sterilized in the disassembled or assembled state and then analyzed for remaining bacteria and spores. Results At all target locations after either a flash sterilization cycle or a standard sterilization cycle, complete eradication of both the vegetative-form and spore-form of G stearothermophilus was achieved. Conclusion This study demonstrates that adequate decontamination of the tested BHs can be achieved after steam sterilization in either the disassembled or assembled state, without an increased risk of infection transmission. PMID:26948131

  18. 29 CFR 1926.1403 - Assembly/Disassembly-selection of manufacturer or employer procedures.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 8 2013-07-01 2013-07-01 false Assembly/Disassembly-selection of manufacturer or employer... CONSTRUCTION Cranes and Derricks in Construction § 1926.1403 Assembly/Disassembly—selection of manufacturer or... applicable to assembly and disassembly, or (b) Employer procedures for assembly and disassembly. Employer...

  19. 29 CFR 1926.1403 - Assembly/Disassembly-selection of manufacturer or employer procedures.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 8 2012-07-01 2012-07-01 false Assembly/Disassembly-selection of manufacturer or employer... CONSTRUCTION Cranes and Derricks in Construction § 1926.1403 Assembly/Disassembly—selection of manufacturer or... applicable to assembly and disassembly, or (b) Employer procedures for assembly and disassembly. Employer...

  20. 29 CFR 1926.1403 - Assembly/Disassembly-selection of manufacturer or employer procedures.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 8 2014-07-01 2014-07-01 false Assembly/Disassembly-selection of manufacturer or employer... CONSTRUCTION Cranes and Derricks in Construction § 1926.1403 Assembly/Disassembly—selection of manufacturer or... applicable to assembly and disassembly, or (b) Employer procedures for assembly and disassembly. Employer...

  1. 29 CFR 1926.1403 - Assembly/Disassembly-selection of manufacturer or employer procedures.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 8 2011-07-01 2011-07-01 false Assembly/Disassembly-selection of manufacturer or employer... CONSTRUCTION Cranes and Derricks in Construction § 1926.1403 Assembly/Disassembly—selection of manufacturer or... applicable to assembly and disassembly, or (b) Employer procedures for assembly and disassembly. Employer...

  2. Filament turnover tunes both force generation and dissipation to control long-range flows in a model actomyosin cortex

    PubMed Central

    McCall, Patrick M.; Gardel, Margaret L.; Munro, Edwin M.

    2017-01-01

    Actomyosin-based cortical flow is a fundamental engine for cellular morphogenesis. Cortical flows are generated by cross-linked networks of actin filaments and myosin motors, in which active stress produced by motor activity is opposed by passive resistance to network deformation. Continuous flow requires local remodeling through crosslink unbinding and and/or filament disassembly. But how local remodeling tunes stress production and dissipation, and how this in turn shapes long range flow, remains poorly understood. Here, we study a computational model for a cross-linked network with active motors based on minimal requirements for production and dissipation of contractile stress: Asymmetric filament compliance, spatial heterogeneity of motor activity, reversible cross-links and filament turnover. We characterize how the production and dissipation of network stress depend, individually, on cross-link dynamics and filament turnover, and how these dependencies combine to determine overall rates of cortical flow. Our analysis predicts that filament turnover is required to maintain active stress against external resistance and steady state flow in response to external stress. Steady state stress increases with filament lifetime up to a characteristic time τm, then decreases with lifetime above τm. Effective viscosity increases with filament lifetime up to a characteristic time τc, and then becomes independent of filament lifetime and sharply dependent on crosslink dynamics. These individual dependencies of active stress and effective viscosity define multiple regimes of steady state flow. In particular our model predicts that when filament lifetimes are shorter than both τc and τm, the dependencies of effective viscosity and steady state stress on filament turnover cancel one another, such that flow speed is insensitive to filament turnover, and shows a simple dependence on motor activity and crosslink dynamics. These results provide a framework for understanding

  3. Roles for the yeast RAD18 and RAD52 DNA repair genes in UV mutagenesis.

    PubMed

    Armstrong, J D; Chadee, D N; Kunz, B A

    1994-11-01

    Experimental evidence indicates that although the Saccharomyces cerevisiae RAD18 and RAD52 genes are not required for nucleotide excision repair, they function in the processing of UV-induced DNA damage in yeast. Conflicting statements regarding the UV mutability of strains deleted for RAD18 prompted us to re-examine the influence of RAD18, and RAD52, on UV mutagenesis. To do so, we characterized mutations induced by UV in SUP4-o, a yeast suppressor tRNA gene. SUP4-o was maintained on a plasmid in isogenic strains that either carried one of two different rad18 deletions (rad18 delta) or had RAD52 disrupted. Both rad18 deletions decreased the frequency of UV-induced SUP4-o mutations to levels close to those for spontaneous mutagenesis in the rad18 delta backgrounds, and prevented a net increase in mutant yield. A detailed analysis of mutations isolated after UV irradiation of one of the rad18 delta strains uncovered little evidence of the specificity features typical for UV mutagenesis in the isogenic repair-proficient (RAD) parent (e.g., predominance of G.C-->A.T transitions). Evidently, UV induction of SUP4-o mutations is highly dependent on the RAD18 gene. Compared to the RAD strain, disruption of RAD52 reduced the frequency and yield of UV mutagenesis by about two-thirds. Closer inspection revealed that 80% of this reduction was due to a decrease in the frequency of G.C-->A.T transitions. In addition, there were differences in the distributions and site specificities of single base-pair substitutions. Thus, RAD52 also participates in UV mutagenesis of a plasmid-borne gene in yeast, but to a lesser extent than RAD18.

  4. Risk of Contamination in Assembled vs Disassembled Instruments in Hip Arthroplasty Surgery.

    PubMed

    Mayer, Ryan R; Bederman, S Samuel; Colin, Vincent M; Berger, Martina M; Cesario, Thomas C; Schwarzkopf, Ran

    2016-08-01

    Periprosthetic joint infection (PJI) is one of the most common causes of revision total hip arthroplasty (THA) and associated with higher costs, prolonged pain, and worse clinical outcomes. Many factors have been linked to increased infection rates, one being the operative equipment and instrumentation used during the surgical procedure. With few arthroplasty instruments designed for complete disassembly and increasingly complex instrument designs, this study seeks to understand the effect that instrument disassembly plays on infection using disassembled and assembled standard femoral broach handles (BHs). Two BHs, not designed for disassembly, were modified and then contaminated in the disassembled state with Geobacillus stearothermophilus vegetative-form bacteria and spores. Using both flash and standard sterilization cycles, the BHs were steam sterilized in the disassembled or assembled state and then analyzed for remaining bacteria and spores. At all target locations after either a flash sterilization cycle or a standard sterilization cycle, complete eradication of both the vegetative-form and spore-form of G stearothermophilus was achieved. This study demonstrates that adequate decontamination of the tested BHs can be achieved after steam sterilization in either the disassembled or assembled state, without an increased risk of infection transmission. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. The Saccharomyces cerevisiae DNA recombination and repair functions of the RAD52 epistasis group inhibit Ty1 transposition.

    PubMed Central

    Rattray, A J; Shafer, B K; Garfinkel, D J

    2000-01-01

    RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or delta) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA. PMID:10655210

  6. Multi-kanban mechanism for personal computer disassembly

    NASA Astrophysics Data System (ADS)

    Udomsawat, Gun; Gupta, Surendra M.; Kamarthi, Sagar V.

    2004-12-01

    The use of personal computers (PCs) continues to increase every year. According to a 1999 figure, 50 percent of all US households owned PCs, a figure that continues to rise every year. With continuous development of sophisticated software, PCs are becoming increasingly powerful. In addition, the price of a PC continues to steadily decline. Furthermore, the typical life of a PC in the workplace is approximately two to three years while in the home it is three to five years. As these PCs become obsolete, they are replaced and the old PCs are disposed of. It is estimated that between 14 and 20 million PCs are retired annually in the US. While 20 to 30% of the units may be resold, the others are discarded. These discards represent a significant potential source of lead for the waste stream. In some communities, waste cathode ray tubes (CRTs) represent the second largest source of lead in the waste stream after vehicular lead acid batteries. PCs are, therefore, not suitable for dumping in landfills. Besides, several components of a PC can be reused and then there are other valuable materials that can also be harvested. And with the advent of product stewardship, product recovery is the best solution for manufacturers. Disassembly line is perhaps the most suitable set up for disassembling PCs. However, planning and scheduling of disassembly on a disassembly line is complicated. In this paper, we discuss some of the complications including product arrival, demand arrival, inventory fluctuation and production control mechanisms. We then show how to overcome them by implementing a multi-kanban mechanism in the PC disassembly line setting. The multi-kanban mechanism relies on dynamic routing of kanbans according to the state of the system. We investigate the multi-kanban mechanism using simulation and demonstrate that this mechanism is superior to the traditional push system in terms of controlling the system"s inventory while maintaining a decent customer service level.

  7. 29 CFR 1926.1406 - Assembly/Disassembly-employer procedures-general requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 8 2012-07-01 2012-07-01 false Assembly/Disassembly-employer procedures-general... CONSTRUCTION Cranes and Derricks in Construction § 1926.1406 Assembly/Disassembly—employer procedures—general requirements. (a) When using employer procedures instead of manufacturer procedures for assembly/disassembly...

  8. 29 CFR 1926.1406 - Assembly/Disassembly-employer procedures-general requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 8 2014-07-01 2014-07-01 false Assembly/Disassembly-employer procedures-general... CONSTRUCTION Cranes and Derricks in Construction § 1926.1406 Assembly/Disassembly—employer procedures—general requirements. (a) When using employer procedures instead of manufacturer procedures for assembly/disassembly...

  9. 29 CFR 1926.1406 - Assembly/Disassembly-employer procedures-general requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 8 2013-07-01 2013-07-01 false Assembly/Disassembly-employer procedures-general... CONSTRUCTION Cranes and Derricks in Construction § 1926.1406 Assembly/Disassembly—employer procedures—general requirements. (a) When using employer procedures instead of manufacturer procedures for assembly/disassembly...

  10. 29 CFR 1926.1406 - Assembly/Disassembly-employer procedures-general requirements.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 8 2011-07-01 2011-07-01 false Assembly/Disassembly-employer procedures-general... CONSTRUCTION Cranes and Derricks in Construction § 1926.1406 Assembly/Disassembly—employer procedures—general requirements. (a) When using employer procedures instead of manufacturer procedures for assembly/disassembly...

  11. The selective autophagy receptor p62 forms a flexible filamentous helical scaffold.

    PubMed

    Ciuffa, Rodolfo; Lamark, Trond; Tarafder, Abul K; Guesdon, Audrey; Rybina, Sofia; Hagen, Wim J H; Johansen, Terje; Sachse, Carsten

    2015-05-05

    The scaffold protein p62/SQSTM1 is involved in protein turnover and signaling and is commonly found in dense protein bodies in eukaryotic cells. In autophagy, p62 acts as a selective autophagy receptor that recognizes and shuttles ubiquitinated proteins to the autophagosome for degradation. The structural organization of p62 in cellular bodies and the interplay of these assemblies with ubiquitin and the autophagic marker LC3 remain to be elucidated. Here, we present a cryo-EM structural analysis of p62. Together with structures of assemblies from the PB1 domain, we show that p62 is organized in flexible polymers with the PB1 domain constituting a helical scaffold. Filamentous p62 is capable of binding LC3 and addition of long ubiquitin chains induces disassembly and shortening of filaments. These studies explain how p62 assemblies provide a large molecular scaffold for the nascent autophagosome and reveal how they can bind ubiquitinated cargo. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Rad-Release

    ScienceCinema

    None

    2017-12-09

    The R&D 100 Award winning Rad-Release Chemical Decontamination Technology is a highly effective (up to 99% removal rate), affordable, patented chemical-foam-clay decontamination process tailored to specific radiological and metal contaminants, which is applicable to a wide variety of substrates. For more information about this project, visit http://www.inl.gov/rd100/2011/rad-release/

  13. MSL-RAD Cruise Operations Concept

    NASA Technical Reports Server (NTRS)

    Brinza, David E.; Zeitlin, Cary; Hassler, Donald; Weigle, Gerald E.; Boettcher, Stephan; Martin, Cesar; Wimmer-Schweingrubber, Robert

    2012-01-01

    The Mars Science Laboratory (MSL) payload includes the Radiation Assessment Detector (RAD) instrument, intended to fully characterize the radiation environment for the MSL mission. The RAD instrument operations concept is intended to reduce impact to spacecraft resources and effort for the MSL operations team. By design, RAD autonomously performs regular science observations without the need for frequent commanding from the Rover Compute Element (RCE). RAD operates with pre-defined "sleep" and "observe" periods, with an adjustable duty cycle for meeting power and data volume constraints during the mission. At the start of a new science observation, RAD performs a pre-observation activity to assess count rates for selected RAD detector elements. Based on this assessment, RAD can enter "solar event" mode, in which instrument parameters (including observation duration) are selected to more effectively characterize the environment. At the end of each observation period, RAD stores a time-tagged, fixed length science data packet in its non-volatile mass memory storage. The operating cadence is defined by adjustable parameters, also stored in non-volatile memory within the instrument. Periodically, the RCE executes an on-board sequence to transfer RAD science data packets from the instrument mass storage to the MSL downlink buffer. Infrequently, the RAD instrument operating configuration is modified by updating internal parameter tables and configuration entries.

  14. 29 CFR 1926.1407 - Power line safety (up to 350 kV)-assembly and disassembly.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 8 2011-07-01 2011-07-01 false Power line safety (up to 350 kV)-assembly and disassembly... Cranes and Derricks in Construction § 1926.1407 Power line safety (up to 350 kV)—assembly and disassembly... area of assembly/disassembly, closer than 20 feet to a power line during the assembly/disassembly...

  15. The effect of sudden server breakdown on the performance of a disassembly line

    NASA Astrophysics Data System (ADS)

    Udomsawat, Gun; Gupta, Surendra M.

    2005-11-01

    Product and material recovery relies on the disassembly process to separate target components or materials from the end-of-life (EOL) products. Disassembly line is especially effective when products in large quantity are disassembled. Unlike an assembly line, a disassembly line is more complex and is subjected to numerous uncertainties including stochastic and multi-level arrivals of component demands, stochastic arrival times for EOL products, and process interruption due to equipment failure. These factors seriously impair the control mechanism in the disassembly line. A common production control mechanism is the traditional push system (TPS). TPS responds to the aforementioned complications by carrying substantial amounts of inventories. An alternative control mechanism is a newly developed multi-kanban pull system (MKS) that relies on dynamic routing of kanbans, which tends to minimize the system's inventories while maintaining demand serviceability. In this paper we explore the impact of sudden breakdown of server on the performance of a disassembly line. We compare the overall performances of the TPS and MKS by considering two scenarios. We present the solution procedure and results for these cases.

  16. BI-RADS update.

    PubMed

    Mercado, Cecilia L

    2014-05-01

    The updated American College of Radiology (ACR) Breast Imaging Reporting and Data System (BI-RADS) has been newly released. This article summarizes the changes and updates that have been made to BI-RADS. The goal of the revised edition continues to be the same: to improve clarification in image interpretation, maintain reporting standardization, and simplify the monitoring of outcomes. The new BI-RADS also introduces new terminology to provide a more universal lexicon across all 3 imaging modalities. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Application of industrial robots in automatic disassembly line of waste LCD displays

    NASA Astrophysics Data System (ADS)

    Wang, Sujuan

    2017-11-01

    In the automatic disassembly line of waste LCD displays, LCD displays are disassembled into plastic shells, metal shields, circuit boards, and LCD panels. Two industrial robots are used to cut metal shields and remove circuit boards in this automatic disassembly line. The functions of these two industrial robots, and the solutions to the critical issues of model selection, the interfaces with PLCs and the workflows were described in detail in this paper.

  18. Connections of intermediate filaments with the nuclear lamina and the cell periphery.

    PubMed

    Katsuma, Y; Swierenga, S H; Marceau, N; French, S W

    1987-01-01

    We investigated the relationship between intermediate filaments (IFs) and other detergent- and nuclease-resistant filamentous structures of cultured liver epithelial cells (T51B cell line) using whole mount unembedded preparations which were sequentially extracted with Triton X-100 and nucleases. Immunogold labelling and stereoscopic observation facilitated the examination of each filamentous structure and their three-dimensional relationships to each other. After solubilizing phospholipid, nucleic acid and soluble cellular protein, the resulting cytoskeleton preparation consisted of a network of cytokeratin and vimentin IFs linked by 3 nm filaments. The IFs were anchored to and determined the position of the nuclear lamina filaments (NLF) network and the centrioles. The NLF was composed of the nuclear lamina filaments measuring 3-6 nm in diameter which radiated from and anchored to the skeleton of the nuclear pores. The IFs located in the nuclear region appeared to be interwoven with the NLF. At the cell surface, the IFs seemed to be attached to the putative actin filament network. They formed a focally interrupted plexus-like structure at the cell periphery. Fragments of vimentin filaments were found among the filamentous network located at the cell surface, and some filaments terminated blindly there.

  19. 2015 RAD Fall Partner Meeting

    EPA Pesticide Factsheets

    This meeting covered the following discussion topics: 2014 RAD partner achievements and trends, national and international efforts to address HFCs, enhancing RAD partner recognition, and communicating the benefits of RAD.

  20. A comparison of different postures for scaffold end-frame disassembly.

    PubMed

    Cutlip, R; Hsiao, H; Garcia, R; Becker, E; Mayeux, B

    2000-10-01

    Overexertion and fall injuries comprise the largest category of nonfatal injuries among scaffold workers. This study was conducted to identify the most favourable scaffold end-frame disassembly techniques and evaluate the associated slip potential by measuring whole-body isometric strength capability and required coefficient of friction (RCOF) to reduce the incidence of injury. Forty-six male construction workers were used to study seven typical postures associated with scaffold end-frame disassembly. An analysis of variance (ANOVA) showed that the isometric forces (334.4-676.3 N) resulting from the seven postures were significantly different (p < 0.05). Three of the disassembly postures resulted in considerable biomechanical stress to workers. The symmetric front-lift method with hand locations at knuckle height would be the most favourable posture; at least 93% of the male construction worker population could handle the end frame with minimum overexertion risk. The static RCOF value resulting from this posture during the disassembly phase was less than 0.2, thus the likelihood of a slip should be low.

  1. Closure of the R Reactor Disassembly Basin at the SRS

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

    Austin, W.E.

    The Facilities Disposition Division (FDD) at the Savannah River Site is engaged in planning the deactivation/closure of three of the site's five reactor disassembly basins. Activities are currently underway at R-Reactor Disassembly Basin and will continue with the P and C disassembly basins. The basins still contain the cooling and shielding water that was present when operations ceased. Low concentrations of radionuclides are present, with tritium, Cs-137, and Sr-90 being the major contributors. Although there is no evidence that any of the basins have leaked, the 50-year-old facilities will eventually contaminate the surrounding groundwaters. The FDD is pursuing a pro-activemore » solution to close the basins in-place and prevent a release to the groundwater. In-situ ion exchange is currently underway at the R-Reactor Disassembly Basin to reduce the Cs and Sr concentrations to levels that would allow release of the treated water to previously used on-site cooling ponds or to prevent ground water impact. The closure will be accomplished under CERCLA.« less

  2. Assembly/Disassembly of DNA-Au Nanoparticles: A Strategy of Intervention

    DOE PAGES

    Lim, I-Im S.; Wang, Lingyan; Chandrachud, Uma; ...

    2008-01-01

    This report describes the viability of a strategy for manipulating the assembly/disassembly processes of DNA-Au nanoparticles by molecular intervention. Using the temperature-induced assembly and disassembly processes of DNAs and gold nanoparticles as a model system, the introduction of a molecular recognition probe is demonstrated to lead to the intervention of the assembly/disassembly processes depending on its specific biorecognition. This process can be detected by monitoring the change in the optical properties of gold nanoparticles and their DNA assemblies. Implications of the preliminary results to exploration of the resulting nanostructures for fine-tuning of the interfacial reactivities in DNA-based bioassays and biomaterialmore » engineering are also discussed.« less

  3. 29 CFR 1926.1404 - Assembly/Disassembly-general requirements (applies to all assembly and disassembly operations).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...'s procedures must be followed. When lifting loads without using outriggers or stabilizers, the manufacturer's procedures must be met regarding truck wedges or screws. (r) Rigging. In addition to following...(o)(3) before assembly/disassembly begins. (5) Boom and jib pick points. The point(s) of attachment...

  4. 29 CFR 1926.1404 - Assembly/Disassembly-general requirements (applies to all assembly and disassembly operations).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...'s procedures must be followed. When lifting loads without using outriggers or stabilizers, the manufacturer's procedures must be met regarding truck wedges or screws. (r) Rigging. In addition to following...(o)(3) before assembly/disassembly begins. (5) Boom and jib pick points. The point(s) of attachment...

  5. 29 CFR 1926.1404 - Assembly/Disassembly-general requirements (applies to all assembly and disassembly operations).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...'s procedures must be followed. When lifting loads without using outriggers or stabilizers, the manufacturer's procedures must be met regarding truck wedges or screws. (r) Rigging. In addition to following...(o)(3) before assembly/disassembly begins. (5) Boom and jib pick points. The point(s) of attachment...

  6. 29 CFR 1926.1404 - Assembly/Disassembly-general requirements (applies to all assembly and disassembly operations).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...'s procedures must be followed. When lifting loads without using outriggers or stabilizers, the manufacturer's procedures must be met regarding truck wedges or screws. (r) Rigging. In addition to following...(o)(3) before assembly/disassembly begins. (5) Boom and jib pick points. The point(s) of attachment...

  7. Low cytoplasmic pH reduces ER-Golgi trafficking and induces disassembly of the Golgi apparatus

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

    Soonthornsit, Jeerawat; Yamaguchi, Yoko; Tamura, Daisuke

    The Golgi apparatus was dramatically disassembled when cells were incubated in a low pH medium. The cis-Golgi disassembled quickly, extended tubules and spread to the periphery of cells within 30 min. In contrast, medial- and trans-Golgi were fragmented in significantly larger structures of smaller numbers at a slower rate and remained largely in structures distinct from the cis-Golgi. Electron microscopy revealed the complete disassembly of the Golgi stack in low pH treated cells. The effect of low pH was reversible; the Golgi apparatus reassembled to form a normal ribbon-like structure within 1–2 h after the addition of a control medium.more » The anterograde ER to Golgi transport and retrograde Golgi to ER transport were both reduced under low pH. Phospholipase A{sub 2} inhibitors (ONO, BEL) effectively suppressed the Golgi disassembly, suggesting that the phospholipase A{sub 2} was involved in the Golgi disassembly. Over-expression of Rab1, 2, 30, 33 and 41 also suppressed the Golgi disassembly under low pH, suggesting that they have protective role against Golgi disassembly. Low pH treatment reduced cytoplasmic pH, but not the luminal pH of the Golgi apparatus, strongly suggesting that reduction of the cytoplasmic pH triggered the Golgi disassembly. Because a lower cytoplasmic pH is induced in physiological or pathological conditions, disassembly of the Golgi apparatus and reduction of vesicular transport through the Golgi apparatus may play important roles in cell physiology and pathology. Furthermore, our findings indicated that low pH treatment can serve as an important tool to analyze the molecular mechanisms that support the structure and function of the Golgi apparatus. - Highlights: • The Golgi apparatus reversibly disassembles by low pH treatment. • The cis-Golgi disassembles quickly generating tubular structures. • Both anterograde and retrograde transport between the ER and the Golgi apparatus are reduced. • Phospholipase A{sub 2

  8. RadTown USA: Basic Information

    MedlinePlus

    ... Burbs Countryside Waterfront Downtown Did you know? RadTown Games Educational Materials Bring RadTown into the classroom with ... Waterfront Downtown Educational Materials RadTown A to Z Games Link to Us Glossary Subscribe to Listserv News ...

  9. Disassembly Properties of Cementitious Finish Joints Using an Induction Heating Method

    PubMed Central

    Ahn, Jaecheol; Noguchi, Takafumi; Kitagaki, Ryoma

    2015-01-01

    Efficient maintenance and upgrading of a building during its lifecycle are difficult because a cementitious finish uses materials and parts with low disassembly properties. Additionally, the reuse and recycling processes during building demolition also present numerous problems from the perspective of environmental technology. In this study, an induction heating (IH) method was used to disassemble cementitious finish joints, which are widely used to join building members and materials. The IH rapidly and selectively heated and weakened these joints. The temperature elevation characteristics of the cementitious joint materials were measured as a function of several resistor types, including wire meshes and punching metals, which are usually used for cementitious finishing. The disassembly properties were evaluated through various tests using conductive resistors in cementitious joints such as mortar. When steel fiber, punching metal, and wire mesh were used as conductive resistors, the cementitious modifiers could be weakened within 30 s. Cementitious joints with conductive resistors also showed complete disassembly with little residual bond strength.

  10. The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage.

    PubMed Central

    Paulovich, A G; Armour, C D; Hartwell, L H

    1998-01-01

    In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication. PMID:9725831

  11. The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage.

    PubMed

    Paulovich, A G; Armour, C D; Hartwell, L H

    1998-09-01

    In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication.

  12. Roles of the checkpoint sensor clamp Rad9-Rad1-Hus1 (911)-complex and the clamp loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe telomere maintenance.

    PubMed

    Khair, Lyne; Chang, Ya-Ting; Subramanian, Lakxmi; Russell, Paul; Nakamura, Toru M

    2010-06-01

    While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1 cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1 cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes.

  13. Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites

    PubMed Central

    Kato, Takamitsu A.; Suzuki, Takehiro; Dohmae, Naoshi; Takizawa, Kazuya; Nakazawa, Yuka; Genet, Matthew D.; Saotome, Mika; Hama, Michio; Nakajima, Nakako Izumi; Hazawa, Masaharu; Tomita, Masanori; Koike, Manabu; Noshiro, Katsuko; Tomiyama, Kenichi; Obara, Chizuka; Gotoh, Takaya; Ui, Ayako; Fujimori, Akira; Nakayama, Fumiaki; Sugasawa, Kaoru; Okayasu, Ryuichi; Tajima, Katsushi

    2018-01-01

    The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism. PMID:29590107

  14. Roles of the Checkpoint Sensor Clamp Rad9-Rad1-Hus1 (911)-Complex and the Clamp Loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe Telomere Maintenance

    PubMed Central

    Khair, Lyne; Chang, Ya-Ting; Subramanian, Lakxmi; Russell, Paul; Nakamura, Toru M.

    2011-01-01

    While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1Δ cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1Δ cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes. PMID:20505337

  15. Germline Mutations in PALB2, BRCA1, and RAD51C, Which Regulate DNA Recombination Repair, in Patients with Gastric Cancer

    PubMed Central

    Sahasrabudhe, Ruta; Lott, Paul; Bohorquez, Mabel; Toal, Ted; Estrada, Ana P.; Suarez, John J.; Brea-Fernández, Alejandro; Cameselle-Teijeiro, José; Pinto, Carla; Ramos, Irma; Mantilla, Alejandra; Prieto, Rodrigo; Corvalan, Alejandro; Norero, Enrique; Alvarez, Carolina; Tapia, Teresa; Carvallo, Pilar; Gonzalez, Luz M.; Cock-Rada, Alicia; Solano, Angela; Neffa, Florencia; Valle, Adriana Della; Yau, Chris; Soares, Gabriela; Borowsky, Alexander; Hu, Nan; He, Li-Ji; Han, Xiao-You; Taylor, Philip R.; Goldstein, Alisa M.; Torres, Javier; Echeverry, Magdalena; Ruiz-Ponte, Clara; Teixeira, Manuel R.; Carvajal Carmona, Luis G.

    2016-01-01

    Up to 10% of cases of gastric cancer are familial, but so far, only mutations in CDH1 have been associated with gastric cancer risk. To identify genetic variants that affect risk for gastric cancer, we collected blood samples from 28 patients with hereditary diffuse gastric cancer (HDGC) not associated with mutations in CDH1 and performed whole-exome sequence analysis. We then analyzed sequences of candidate genes in 333 independent HDGC and non-HDGC cases. We identified 11 cases with mutations in PALB2, BRCA1, or RAD51C genes, which regulate homologous DNA recombination. We found these mutations in 2 of 31 patients with HDGC (6.5%) and 9 of 331 patients with sporadic gastric cancer (2.8%). Most of these mutations had been previously associated with other types of tumors and partially co-segregated with gastric cancer in our study. Tumors that developed in patients with these mutations had a mutation signature associated with somatic homologous recombination deficiency. Our findings indicate that defects in homologous recombination increase risk for gastric cancer. PMID:28024868

  16. 40 CFR 1065.1109 - Post-test sampler disassembly and sample extraction.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 33 2014-07-01 2014-07-01 false Post-test sampler disassembly and... Semi-Volatile Organic Compounds § 1065.1109 Post-test sampler disassembly and sample extraction. This... environment as follows after the test: (1) Remove the PM filter, PUF plugs, and all the XAD-2 from the...

  17. Cdc13 prevents telomere uncapping and Rad50-dependent homologous recombination

    PubMed Central

    Grandin, Nathalie; Damon, Christelle; Charbonneau, Michel

    2001-01-01

    Cdc13 performs an essential function in telomere end protection in budding yeast. Here, we analyze the consequences on telomere dynamics of cdc13-induced telomeric DNA damage in proliferating cells. Checkpoint-deficient cdc13-1 cells accumulated DNA damage and eventually senesced. However, these telomerase-proficient cells could survive by using homologous recombination but, contrary to telomerase-deficient cells, did so without prior telomere shortening. Strikingly, homologous recombination in cdc13-1 mec3, as well as in telomerase-deficient cdc13-1 cells, which were Rad52- and Rad50-dependent but Rad51-independent, exclusively amplified the TG1–3 repeats. This argues that not only short telomeres are substrates for type II recombination. The Cdc13-1 mutant protein harbored a defect in its association with Stn1 and Ten1 but also an additional, unknown, defect that could not be cured by expressing a Cdc13-1– Ten1–Stn1 fusion. We propose that Cdc13 prevents telomere uncapping and inhibits recombination between telomeric sequences through a pathway distinct from and complementary to that used by telomerase. PMID:11689452

  18. Capillarity-induced disassembly of virions in carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Fan, Xiaobin; Barclay, J. Elaine; Peng, Wenchao; Li, Yang; Li, Xianyu; Zhang, Guoliang; Evans, David J.; Zhang, Fengbao

    2008-04-01

    Studying the transport and fate of viruses through nanochannels is of great importance. By using the nanochannel of a carbon nanotube (CNT) as an ideal model, we evaluated the possibility of capillarity-induced viral transport through a closely fitting nanochannel and explored the mechanisms involved. It is shown both experimentally and theoretically that Cowpea mosaic virus can enter CNTs by capillarity. However, when introduced into a nanotube the protein capsid may disassemble. During the initial capillary filling stage, anomalous needle-shaped high pressure exists in the centre of the nanotube's entrance. This high pressure, combining with the significant negative pressure within the nanotube, may account for the disassembly of the virions.

  19. Low cytoplasmic pH reduces ER-Golgi trafficking and induces disassembly of the Golgi apparatus.

    PubMed

    Soonthornsit, Jeerawat; Yamaguchi, Yoko; Tamura, Daisuke; Ishida, Ryuichi; Nakakoji, Yoko; Osako, Shiho; Yamamoto, Akitsugu; Nakamura, Nobuhiro

    2014-11-01

    The Golgi apparatus was dramatically disassembled when cells were incubated in a low pH medium. The cis-Golgi disassembled quickly, extended tubules and spread to the periphery of cells within 30 min. In contrast, medial- and trans-Golgi were fragmented in significantly larger structures of smaller numbers at a slower rate and remained largely in structures distinct from the cis-Golgi. Electron microscopy revealed the complete disassembly of the Golgi stack in low pH treated cells. The effect of low pH was reversible; the Golgi apparatus reassembled to form a normal ribbon-like structure within 1-2h after the addition of a control medium. The anterograde ER to Golgi transport and retrograde Golgi to ER transport were both reduced under low pH. Phospholipase A2 inhibitors (ONO, BEL) effectively suppressed the Golgi disassembly, suggesting that the phospholipase A2 was involved in the Golgi disassembly. Over-expression of Rab1, 2, 30, 33 and 41 also suppressed the Golgi disassembly under low pH, suggesting that they have protective role against Golgi disassembly. Low pH treatment reduced cytoplasmic pH, but not the luminal pH of the Golgi apparatus, strongly suggesting that reduction of the cytoplasmic pH triggered the Golgi disassembly. Because a lower cytoplasmic pH is induced in physiological or pathological conditions, disassembly of the Golgi apparatus and reduction of vesicular transport through the Golgi apparatus may play important roles in cell physiology and pathology. Furthermore, our findings indicated that low pH treatment can serve as an important tool to analyze the molecular mechanisms that support the structure and function of the Golgi apparatus. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Foraging traits modulate stingless bee community disassembly under forest loss.

    PubMed

    Lichtenberg, Elinor M; Mendenhall, Chase D; Brosi, Berry

    2017-10-01

    Anthropogenic land use change is an important driver of impacts to biological communities and the ecosystem services they provide. Pollination is one ecosystem service that may be threatened by community disassembly. Relatively little is known about changes in bee community composition in the tropics, where pollination limitation is most severe and land use change is rapid. Understanding how anthropogenic changes alter community composition and functioning has been hampered by high variability in responses of individual species. Trait-based approaches, however, are emerging as a potential method for understanding responses of ecologically similar species to global change. We studied how communities of tropical, eusocial stingless bees (Apidae: Meliponini) disassemble when forest is lost. These bees are vital tropical pollinators that exhibit high trait diversity, but are under considerable threat from human activities. We compared functional traits of stingless bee species found in pastures surrounded by differing amounts of forest in an extensively deforested landscape in southern Costa Rica. Our results suggest that foraging traits modulate competitive interactions that underlie community disassembly patterns. In contrast to both theoretical predictions and temperate bee communities, we found that stingless bee species with the widest diet breadths were less likely to persist in sites with less forest. These wide-diet-breadth species also tend to be solitary foragers, and are competitively subordinate to group-foraging stingless bee species. Thus, displacement by dominant, group-foraging species may make subordinate species more dependent on the larger or more diversified resource pool that natural habitats offer. We also found that traits that may reduce reliance on trees-nesting in the ground or inside nests of other species-correlated with persistence in highly deforested landscapes. The functional trait perspective we employed enabled capturing community

  1. Effects of reactive oxygen species on cellular wall disassembly of banana fruit during ripening.

    PubMed

    Cheng, Guiping; Duan, Xuewu; Shi, John; Lu, Wangjin; Luo, Yunbo; Jiang, Weibo; Jiang, Yueming

    2008-07-15

    Fruit softening is generally attributed to cell wall disassembly. Experiments were conducted to investigate effects of various reactive oxygen species (ROS) on in vitro cellular wall disassembly of harvested banana fruit. The alcohol-extracted insoluble residue (AEIR) was obtained from the pulp tissues of banana fruit at various ripening stages and then used to examine the disassembly of cellular wall polysaccharides in the presence of superoxide anion (O2(-)), hydrogen peroxide (H2O2) or hydroxyl radical (OH) and their scavengers. The presence of OH accelerated significantly disassembly of cellular wall polysaccharides in terms of the increase in contents of total sugars released and uronic acid, and the decrease in molecular mass of soluble polysaccharides, using gel permeation chromatography. However, the treatment with H2O2 or O2(-) showed no significant effect on the disassembly of cellular wall polysaccharides. Furthermore, the degradation of the de-esterified AEIR was more susceptible to OH attack than the esterified AEIR. In addition, the effect of OH could be inhibited in the presence of OH scavenger. This study suggests that disassembly of cellular wall polysaccharides could be initiated by OH as the solublisation of the polysaccharides increased, which, in turn, accelerated fruit softening. Copyright © 2008 Elsevier Ltd. All rights reserved.

  2. Meiotic Clade AAA ATPases: Protein Polymer Disassembly Machines.

    PubMed

    Monroe, Nicole; Hill, Christopher P

    2016-05-08

    Meiotic clade AAA ATPases (ATPases associated with diverse cellular activities), which were initially grouped on the basis of phylogenetic classification of their AAA ATPase cassette, include four relatively well characterized family members, Vps4, spastin, katanin and fidgetin. These enzymes all function to disassemble specific polymeric protein structures, with Vps4 disassembling the ESCRT-III polymers that are central to the many membrane-remodeling activities of the ESCRT (endosomal sorting complexes required for transport) pathway and spastin, katanin p60 and fidgetin affecting multiple aspects of cellular dynamics by severing microtubules. They share a common domain architecture that features an N-terminal MIT (microtubule interacting and trafficking) domain followed by a single AAA ATPase cassette. Meiotic clade AAA ATPases function as hexamers that can cycle between the active assembly and inactive monomers/dimers in a regulated process, and they appear to disassemble their polymeric substrates by translocating subunits through the central pore of their hexameric ring. Recent studies with Vps4 have shown that nucleotide-induced asymmetry is a requirement for substrate binding to the pore loops and that recruitment to the protein lattice via MIT domains also relieves autoinhibition and primes the AAA ATPase cassettes for substrate binding. The most striking, unifying feature of meiotic clade AAA ATPases may be their MIT domain, which is a module that is found in a wide variety of proteins that localize to ESCRT-III polymers. Spastin also displays an adjacent microtubule binding sequence, and the presence of both ESCRT-III and microtubule binding elements may underlie the recent findings that the ESCRT-III disassembly function of Vps4 and the microtubule-severing function of spastin, as well as potentially katanin and fidgetin, are highly coordinated. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Rare adipose disorders (RADs) masquerading as obesity

    PubMed Central

    Herbst, Karen L

    2012-01-01

    Rare adipose disorders (RADs) including multiple symmetric lipomatosis (MSL), lipedema and Dercum's disease (DD) may be misdiagnosed as obesity. Lifestyle changes, such as reduced caloric intake and increased physical activity are standard care for obesity. Although lifestyle changes and bariatric surgery work effectively for the obesity component of RADs, these treatments do not routinely reduce the abnormal subcutaneous adipose tissue (SAT) of RADs. RAD SAT likely results from the growth of a brown stem cell population with secondary lymphatic dysfunction in MSL, or by primary vascular and lymphatic dysfunction in lipedema and DD. People with RADs do not lose SAT from caloric limitation and increased energy expenditure alone. In order to improve recognition of RADs apart from obesity, the diagnostic criteria, histology and pathophysiology of RADs are presented and contrasted to familial partial lipodystrophies, acquired partial lipodystrophies and obesity with which they may be confused. Treatment recommendations focus on evidence-based data and include lymphatic decongestive therapy, medications and supplements that support loss of RAD SAT. Associated RAD conditions including depression, anxiety and pain will improve as healthcare providers learn to identify and adopt alternative treatment regimens for the abnormal SAT component of RADs. Effective dietary and exercise regimens are needed in RAD populations to improve quality of life and construct advanced treatment regimens for future generations. PMID:22301856

  4. Differentiation of prostatitis and prostate cancer using the Prostate Imaging-Reporting and Data System (PI-RADS).

    PubMed

    Meier-Schroers, Michael; Kukuk, Guido; Wolter, Karsten; Decker, Georges; Fischer, Stefan; Marx, Christian; Traeber, Frank; Sprinkart, Alois Martin; Block, Wolfgang; Schild, Hans Heinz; Willinek, Winfried

    2016-07-01

    To determine if prostate cancer (PCa) and prostatitis can be differentiated by using PI-RADS. 3T MR images of 68 patients with 85 cancer suspicious lesions were analyzed. The findings were correlated with histopathology. T2w imaging (T2WI), diffusion weighted imaging (DWI), dynamic contrast enhancement (DCE), and MR-Spectroscopy (MRS) were acquired. Every lesion was given a single PI-RADS score for each parameter, as well as a sum score and a PI-RADS v2 score. Furthermore, T2-morphology, ADC-value, perfusion type, citrate/choline-level, and localization were evaluated. 44 of 85 lesions showed PCa (51.8%), 21 chronic prostatitis (24.7%), and 20 other benign tissue such as hyperplasia or fibromuscular tissue (23.5%). The single PI-RADS score for T2WI, DWI, DCE, as well as the aggregated score including and not including MRS, and the PI-RADS v2-score were all significantly higher for PCa than for prostatitis or other tissue (p<0.001). The single PI-RADS score for MRS and the PI-RADS sum score including MRS were significantly higher for prostatitis than for other tissue (p=0.029 and p=0.020), whereas the other parameters were not different. Prostatitis usually presented borderline pathological PI-RADS scores, showed restricted diffusion with ADC≥900mm(2)/s in 100% of cases, was more often indistinctly hypointense on T2WI (66.7%), and localized in the transitional zone (57.1%). An ADC≥900mm(2)/s achieved the highest predictive value for prostatitis (AUC=0.859). Prostatitis can be differentiated from PCa using PI-RADS, since all available parameters are more distinct in cases of cancer. However, there is significant overlap between prostatitis and other benign findings, thus PI-RADS is only suitable to a limited extent for the primary assessment of prostatitis. Restricted diffusion with ADC≥900mm(2)/s is believed to be a good indicator for prostatitis. MRS can help to distinguish between prostatitis and other tissue. Copyright © 2016 Elsevier Ireland Ltd. All

  5. Contribution of actin filaments to the global compressive properties of fibroblasts.

    PubMed

    Ujihara, Yoshihiro; Nakamura, Masanori; Miyazaki, Hiroshi; Wada, Shigeo

    2012-10-01

    Actin filaments are often regarded as tension-bearing components. Here, we examined the effects of actin filaments on global compressive properties of cells experimentally and numerically. Fibroblasts were harvested from the patellar tendon of a mature Japanese white rabbit and treated with cytochalasin D to depolymerize the actin filaments. Intact cells and cells with disrupted actin filaments were subjected to the compressive tests. Each floating cell was held between the cantilever and compressive plates and compressed by moving the compressive plate with a linear actuator to obtain a load-deformation curve under quasi-static conditions. The experimental results demonstrated that the initial stiffness of a cell with disrupted actin filaments decreased by 51%. After the experiments, we simulated the compressive test of cells with/without bundles of actin filaments. A bundle of actin filaments was modeled as a tension-bearing component that generates a force based on Hooke's law only when it was elongated. By contrast, if it was shortened, it was assumed to exert no force. The computational results revealed that the alignment of bundles of actin filaments significantly affected the cell stiffness. In addition, the passive reorientation of bundles of actin filaments perpendicular to the compression induced an increase in the resistance to the vertical elongation of a cell and thereby increased the cell stiffness. These results clearly indicated that bundles of actin filaments contribute to the compressive properties of a cell, even if they are tension-bearing components. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. RAD50 germline mutations are associated with poor survival in BRCA1/2-negative breast cancer patients.

    PubMed

    Fan, Cong; Zhang, Juan; Ouyang, Tao; Li, Jinfeng; Wang, Tianfeng; Fan, Zhaoqing; Fan, Tie; Lin, Benyao; Xie, Yuntao

    2018-05-04

    RAD50 is a highly conserved DNA double-strand break (DSB) repair gene. However, the associations between RAD50 germline mutations and the survival and risk of breast cancer have not been fully elucidated. Here, we aimed to investigate the clinical impact of RAD50 germline mutations in a large cohort of unselected breast cancer patients. In this study, RAD50 germline mutations were determined using next-generation sequencing in 7657 consecutive unselected breast cancer patients without BRCA1/2 mutations. We also screened for RAD50 recurrent mutations (L719fs, K994fs, and H1269fs) in 5000 healthy controls using Sanger sequencing. We found that 26 out of 7657 (0.34%) patients had RAD50 pathogenic mutations, and 16 patients carried one of the three recurrent mutations (L719fs, n=6 cases; K994fs, n=5 cases; and H1269fs, n=5 cases); the recurrent mutation rate was 0.21%. The frequency of the three recurrent mutations in the 5000 healthy controls was 0.18% (9/5000). These mutations did not confer an increased risk of breast cancer in the studied patients [odds ratios (OR), 1.16; 95% confidence interval (CI), 0.51-2.63; P = 0.72]. Nevertheless, multivariate analysis revealed that RAD50 pathogenic mutations were an independent unfavourable predictor of recurrence-free survival (RFS) [adjusted hazard ratio (HR) 2.66; 95% CI, 1.18-5.98; P=0.018] and disease-specific survival (DSS) (adjusted HR 4.36; 95% CI, 1.58-12.03; P=0.004) in the entire study cohort. Our study suggested that RAD50 germline mutations are not associated with an increased risk of breast cancer, but patients with RAD50 germline mutations have unfavourable survival compared with patients without these mutations. This article is protected by copyright. All rights reserved. © 2018 UICC.

  7. E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6

    PubMed Central

    Hibbert, Richard G.; Huang, Anding; Boelens, Rolf; Sixma, Titia K.

    2011-01-01

    In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for “error prone” DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation. This activity is prevented during PCNA ubiquitination by the interaction of Rad6 with E3 enzyme Rad18. Using NMR and X-ray crystallography we show that the R6BD of Rad18 inhibits this activity by competing with ubiquitin for a noncovalent “backside” binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process. PMID:21422291

  8. Germline Mutations in PALB2, BRCA1, and RAD51C, Which Regulate DNA Recombination Repair, in Patients With Gastric Cancer.

    PubMed

    Sahasrabudhe, Ruta; Lott, Paul; Bohorquez, Mabel; Toal, Ted; Estrada, Ana P; Suarez, John J; Brea-Fernández, Alejandro; Cameselle-Teijeiro, José; Pinto, Carla; Ramos, Irma; Mantilla, Alejandra; Prieto, Rodrigo; Corvalan, Alejandro; Norero, Enrique; Alvarez, Carolina; Tapia, Teresa; Carvallo, Pilar; Gonzalez, Luz M; Cock-Rada, Alicia; Solano, Angela; Neffa, Florencia; Della Valle, Adriana; Yau, Chris; Soares, Gabriela; Borowsky, Alexander; Hu, Nan; He, Li-Ji; Han, Xiao-You; Taylor, Philip R; Goldstein, Alisa M; Torres, Javier; Echeverry, Magdalena; Ruiz-Ponte, Clara; Teixeira, Manuel R; Carvajal-Carmona, Luis G

    2017-04-01

    Up to 10% of cases of gastric cancer are familial, but so far, only mutations in CDH1 have been associated with gastric cancer risk. To identify genetic variants that affect risk for gastric cancer, we collected blood samples from 28 patients with hereditary diffuse gastric cancer (HDGC) not associated with mutations in CDH1 and performed whole-exome sequence analysis. We then analyzed sequences of candidate genes in 333 independent HDGC and non-HDGC cases. We identified 11 cases with mutations in PALB2, BRCA1, or RAD51C genes, which regulate homologous DNA recombination. We found these mutations in 2 of 31 patients with HDGC (6.5%) and 9 of 331 patients with sporadic gastric cancer (2.8%). Most of these mutations had been previously associated with other types of tumors and partially co-segregated with gastric cancer in our study. Tumors that developed in patients with these mutations had a mutation signature associated with somatic homologous recombination deficiency. Our findings indicate that defects in homologous recombination increase risk for gastric cancer. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

  9. The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination.

    PubMed

    Eppink, Berina; Tafel, Agnieszka A; Hanada, Katsuhiro; van Drunen, Ellen; Hickson, Ian D; Essers, Jeroen; Kanaar, Roland

    2011-11-10

    Ultraviolet (UV) radiation-induced DNA lesions can be efficiently repaired by nucleotide excision repair (NER). However, NER is less effective during replication of UV-damaged chromosomes. In contrast, translesion DNA synthesis (TLS) and homologous recombination (HR) are capable of dealing with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES cells lacking RAD54 are not UV sensitive. Here we show that the requirement for mammalian RAD54 is masked by active NER. By genetically inactivating NER and HR through disruption of the Xpa and Rad54 genes, respectively, we demonstrate the contribution of HR to chromosomal integrity upon UV irradiation. We demonstrate using chromosome fiber analysis at the individual replication fork level, that HR activity is important for the restart of DNA replication after induction of DNA damage by UV-light in NER-deficient cells. Furthermore, our data reveal RAD54-dependent and -independent contributions of HR to the cellular sensitivity to UV-light, and they uncover that RAD54 can compensate for the loss of TLS polymerase η with regard to UV-light sensitivity. In conclusion, we show that HR is important for the progression of UV-stalled replication forks in ES cells, and that protection of the fork is an interplay between HR and TLS. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.

    PubMed

    Mbantenkhu, MacMillan; Wang, Xiaowen; Nardozzi, Jonathan D; Wilkens, Stephan; Hoffman, Elizabeth; Patel, Anamika; Cosgrove, Michael S; Chen, Xin Jie

    2011-12-09

    Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ∼14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.

  11. Low-dose irradiation promotes Rad51 expression by down-regulating miR-193b-3p in hepatocytes

    NASA Astrophysics Data System (ADS)

    Lee, Eon-Seok; Won, Yeo Jin; Kim, Byoung-Chul; Park, Daeui; Bae, Jin-Han; Park, Seong-Joon; Noh, Sung Jin; Kang, Yeong-Rok; Choi, Si Ho; Yoon, Je-Hyun; Heo, Kyu; Yang, Kwangmo; Son, Tae Gen

    2016-05-01

    Current evidence indicates that there is a relationship between microRNA (miRNA)-mediated gene silencing and low-dose irradiation (LDIR) responses. Here, alterations of miRNA expression in response to LDIR exposure in male BALB/c mice and three different types of hepatocytes were investigated. The miRNome of the LDIR-exposed mouse spleens (0.01 Gy, 6.5 mGy/h) was analyzed, and the expression of miRNA and mRNA was validated by qRT-PCR. Western blotting, chromatin immunoprecipitation (ChIP), and luciferase assays were also performed to evaluate the interaction between miRNAs and their target genes and to gain insight into the regulation of miRNA expression. The expression of miRNA-193b-3p was down-regulated in the mouse spleen and liver and in various hepatocytes (NCTC, Hepa, and HepG2 cell lines) in response to LDIR. The down-regulation of miR-193b-3p expression was caused by histone deacetylation on the miR-193b-3p promoter in the HepG2 cells irradiated with 0.01 Gy. However, the alteration of histone deacetylation and miR-193b-3p and Rad51 expression in response to LDIR was restored by pretreatment with N-acetyl-cyctein. In conclusion, we provide evidence that miRNA responses to LDIR include the modulation of cellular stress responses and repair mechanisms.

  12. Polymer–Peptide Conjugates Disassemble Amyloid β Fibrils in a Molecular-Weight Dependent Manner

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

    Song, Yang; Moore, Edwin G.; Guo, Yanshu

    Amyloid aggregation and deposition are associated with many intractable human diseases. Although the inhibition of amyloid protein aggregation has been well-studied, the disaggregation and dissolution of existing amyloid fibrils is less known. Taking a fibrillar assembly of amyloid β (Aβ) peptide as the model system, in this paper we report multivalent polymer–peptide conjugates (mPPCs) that disassemble preformed Aβ fibrils into dispersible sub-100 nm structures. Atomic force microscopy and dynamic light scattering studies show that the disassembly rate of preformed Aβ fibrils is controlled by the molecular weight of mPPCs. Rate equations on fibril disappearance are deduced from a simple model,more » which indicate that the disassembly reaction is first-order in the concentration of Aβ fibrils and a pseudo-first-order reaction in the concentration of peptide moieties on mPPCs, respectively. We eliminate the possibility that the disassembly occurs by the association between mPPCs and Aβ monomer/oligomers based on circular dichroism and Thioflavin T fluorescence assays. It is mostly likely that the mPPCs disassemble Aβ fibrils through a direct interaction. Finally, the mPPCs may thus offer a general macromolecular design concept that breaks down existing amyloid fibrils in a predictable fashion.« less

  13. Polymer–Peptide Conjugates Disassemble Amyloid β Fibrils in a Molecular-Weight Dependent Manner

    DOE PAGES

    Song, Yang; Moore, Edwin G.; Guo, Yanshu; ...

    2017-03-14

    Amyloid aggregation and deposition are associated with many intractable human diseases. Although the inhibition of amyloid protein aggregation has been well-studied, the disaggregation and dissolution of existing amyloid fibrils is less known. Taking a fibrillar assembly of amyloid β (Aβ) peptide as the model system, in this paper we report multivalent polymer–peptide conjugates (mPPCs) that disassemble preformed Aβ fibrils into dispersible sub-100 nm structures. Atomic force microscopy and dynamic light scattering studies show that the disassembly rate of preformed Aβ fibrils is controlled by the molecular weight of mPPCs. Rate equations on fibril disappearance are deduced from a simple model,more » which indicate that the disassembly reaction is first-order in the concentration of Aβ fibrils and a pseudo-first-order reaction in the concentration of peptide moieties on mPPCs, respectively. We eliminate the possibility that the disassembly occurs by the association between mPPCs and Aβ monomer/oligomers based on circular dichroism and Thioflavin T fluorescence assays. It is mostly likely that the mPPCs disassemble Aβ fibrils through a direct interaction. Finally, the mPPCs may thus offer a general macromolecular design concept that breaks down existing amyloid fibrils in a predictable fashion.« less

  14. Microinterventional endocapsular nucleus disassembly: novel technique and results of first-in-human randomised controlled study.

    PubMed

    Ianchulev, Tsontcho; Chang, David F; Koo, Edward; MacDonald, Susan; Calvo, Ernesto; Tyson, Farrell Toby; Vasquez, Andrea; Ahmed, Iqbal Ike K

    2018-04-18

    To assess the safety and efficacy of microinterventional endocapsular nuclear fragmentation in moderate to severe cataracts. This was a prospective single-masked multisurgeon interventional randomised controlled trial (ClinicalTrials.gov NCT02843594) where 101 eyes of 101 subjects with grade 3-4+ nuclear cataracts were randomised to torsional phacoemulsification alone (controls) or torsional phacoemulsification with adjunctive endocapsular nuclear fragmentation using a manual microinterventional nitinol filament loop device (miLOOP group). Outcome measures were phacoemulsification efficiency as measured by ultrasound energy (cumulative dispersed energy (CDE) units) and fluidics requirements (total irrigation fluid used) as well as incidence of intraoperative and postoperative complications. Only high-grade advanced cataracts were enrolled with more than 85% of eyes with baseline best corrected visual acuity (BCVA) of 20/200 or worse in either group. Mean CDE was 53% higher in controls (32.8±24.9 vs 21.4±13.1 with miLOOP assistance) (p=0.004). Endothelial cell loss after surgery was low and similar between groups (7-8%, p=0.561) One-month BCVA averaged 20/27 Snellen in miLOOP eyes and 20/24 in controls. No direct complications were caused by the miLOOP. In two cases, capsular tears occurred during IOL implantation and in all remaining cases during phacoemulsification, with none occurring during the miLOOP nucleus disassembly part of the procedure. Microinterventional endocapsular fragmentation with the manual, disposable miLOOP device achieved consistent, ultrasound-free, full-thickness nucleus disassembly and significantly improved overall phaco efficiency in advanced cataracts. NCT02843594. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  15. Large amplitude oscillatory motion along a solar filament

    NASA Astrophysics Data System (ADS)

    Vršnak, B.; Veronig, A. M.; Thalmann, J. K.; Žic, T.

    2007-08-01

    Context: Large amplitude oscillations of solar filaments is a phenomenon that has been known for more than half a century. Recently, a new mode of oscillations, characterized by periodical plasma motions along the filament axis, was discovered. Aims: We analyze such an event, recorded on 23 January 2002 in Big Bear Solar Observatory Hα filtergrams, to infer the triggering mechanism and the nature of the restoring force. Methods: Motion along the filament axis of a distinct buldge-like feature was traced, to quantify the kinematics of the oscillatory motion. The data were fitted by a damped sine function to estimate the basic parameters of the oscillations. To identify the triggering mechanism, morphological changes in the vicinity of the filament were analyzed. Results: The observed oscillations of the plasma along the filament were characterized by an initial displacement of 24 Mm, an initial velocity amplitude of 51 km s-1, a period of 50 min, and a damping time of 115 min. We interpret the trigger in terms of poloidal magnetic flux injection by magnetic reconnection at one of the filament legs. The restoring force is caused by the magnetic pressure gradient along the filament axis. The period of oscillations, derived from the linearized equation of motion (harmonic oscillator) can be expressed as P=π√{2}L/v_Aϕ≈4.4L/v_Aϕ, where v_Aϕ =Bϕ0/√μ_0ρ represents the Alfvén speed based on the equilibrium poloidal field Bϕ0. Conclusions: Combination of our measurements with some previous observations of the same kind of oscillations shows good agreement with the proposed interpretation. Movie to Fig. 1 is only available in electronic form at http://www.aanda.org

  16. Interactome disassembly during apoptosis occurs independent of caspase cleavage.

    PubMed

    Scott, Nichollas E; Rogers, Lindsay D; Prudova, Anna; Brown, Nat F; Fortelny, Nikolaus; Overall, Christopher M; Foster, Leonard J

    2017-01-12

    Protein-protein interaction networks (interactomes) define the functionality of all biological systems. In apoptosis, proteolysis by caspases is thought to initiate disassembly of protein complexes and cell death. Here we used a quantitative proteomics approach, protein correlation profiling (PCP), to explore changes in cytoplasmic and mitochondrial interactomes in response to apoptosis initiation as a function of caspase activity. We measured the response to initiation of Fas-mediated apoptosis in 17,991 interactions among 2,779 proteins, comprising the largest dynamic interactome to date. The majority of interactions were unaffected early in apoptosis, but multiple complexes containing known caspase targets were disassembled. Nonetheless, proteome-wide analysis of proteolytic processing by terminal amine isotopic labeling of substrates (TAILS) revealed little correlation between proteolytic and interactome changes. Our findings show that, in apoptosis, significant interactome alterations occur before and independently of caspase activity. Thus, apoptosis initiation includes a tight program of interactome rearrangement, leading to disassembly of relatively few, select complexes. These early interactome alterations occur independently of cleavage of these protein by caspases. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  17. Coordination of Rad1-Rad10 interactions with Msh2-Msh3, Saw1 and RPA is essential for functional 3' non-homologous tail removal.

    PubMed

    Eichmiller, Robin; Medina-Rivera, Melisa; DeSanto, Rachel; Minca, Eugen; Kim, Christopher; Holland, Cory; Seol, Ja-Hwan; Schmit, Megan; Oramus, Diane; Smith, Jessica; Gallardo, Ignacio F; Finkelstein, Ilya J; Lee, Sang Eun; Surtees, Jennifer A

    2018-06-01

    Double strand DNA break repair (DSBR) comprises multiple pathways. A subset of DSBR pathways, including single strand annealing, involve intermediates with 3' non-homologous tails that must be removed to complete repair. In Saccharomyces cerevisiae, Rad1-Rad10 is the structure-specific endonuclease that cleaves the tails in 3' non-homologous tail removal (3' NHTR). Rad1-Rad10 is also an essential component of the nucleotide excision repair (NER) pathway. In both cases, Rad1-Rad10 requires protein partners for recruitment to the relevant DNA intermediate. Msh2-Msh3 and Saw1 recruit Rad1-Rad10 in 3' NHTR; Rad14 recruits Rad1-Rad10 in NER. We created two rad1 separation-of-function alleles, rad1R203A,K205A and rad1R218A; both are defective in 3' NHTR but functional in NER. In vitro, rad1R203A,K205A was impaired at multiple steps in 3' NHTR. The rad1R218A in vivo phenotype resembles that of msh2- or msh3-deleted cells; recruitment of rad1R218A-Rad10 to recombination intermediates is defective. Interactions among rad1R218A-Rad10 and Msh2-Msh3 and Saw1 are altered and rad1R218A-Rad10 interactions with RPA are compromised. We propose a model in which Rad1-Rad10 is recruited and positioned at the recombination intermediate through interactions, between Saw1 and DNA, Rad1-Rad10 and Msh2-Msh3, Saw1 and Msh2-Msh3 and Rad1-Rad10 and RPA. When any of these interactions is altered, 3' NHTR is impaired.

  18. Three new genetic loci (R1210C in CFH, variants in COL8A1 and RAD51B) are independently related to progression to advanced macular degeneration.

    PubMed

    Seddon, Johanna M; Reynolds, Robyn; Yu, Yi; Rosner, Bernard

    2014-01-01

    To assess the independent impact of new genetic variants on conversion to advanced stages of AMD, controlling for established risk factors, and to determine the contribution of genes in predictive models. In this prospective longitudinal study of 2765 individuals, 777 subjects progressed to neovascular disease (NV) or geographic atrophy (GA) in either eye over 12 years. Recently reported genetic loci were assessed for their independent effects on incident advanced AMD after controlling for 6 established loci in 5 genes, and demographic, behavioral, and macular characteristics. New variants which remained significantly related to progression were then added to a final multivariate model to assess their independent effects. The contribution of genes to risk models was assessed using reclassification tables by determining risk within cross-classified quintiles for alternative models. THREE NEW GENETIC VARIANTS WERE SIGNIFICANTLY RELATED TO PROGRESSION: rare variant R1210C in CFH (hazard ratio (HR) 2.5, 95% confidence interval [CI] 1.2-5.3, P = 0.01), and common variants in genes COL8A1 (HR 2.0, 95% CI 1.1-3.5, P = 0.02) and RAD51B (HR 0.8, 95% CI 0.60-0.97, P = 0.03). The area under the curve statistic (AUC) was significantly higher for the 9 gene model (.884) vs the 0 gene model (.873), P = .01. AUC's for the 9 vs 6 gene models were not significantly different, but reclassification analyses indicated significant added information for more genes, with adjusted odds ratios (OR) for progression within 5 years per one quintile increase in risk score of 2.7, P<0.001 for the 9 vs 6 loci model, and OR 3.5, P<0.001 for the 9 vs. 0 gene model. Similar results were seen for NV and GA. Rare variant CFH R1210C and common variants in COL8A1 and RAD51B plus six genes in previous models contribute additional predictive information for advanced AMD beyond macular and behavioral phenotypes.

  19. Rad GTPase Deficiency Leads to Cardiac Hypertrophy

    PubMed Central

    Tseng, Yu-Hua; Xie, Chang-Qing; Ilany, Jacob; Brüning, Jens C.; Sun, Zhongcui; Zhu, Xiaojun; Cui, Taixing; Youker, Keith A.; Yang, Qinglin; Day, Sharlene M.; Kahn, C. Ronald; Chen, Y. Eugene

    2014-01-01

    Background Rad (Ras associated with diabetes) GTPase is the prototypic member of a subfamily of Ras-related small G proteins. The aim of the present study was to define whether Rad plays an important role in mediating cardiac hypertrophy. Methods and Results We document for the first time that levels of Rad mRNA and protein were decreased significantly in human failing hearts (n=10) compared with normal hearts (n=3; P<0.01). Similarly, Rad expression was decreased significantly in cardiac hypertrophy induced by pressure overload and in cultured cardiomyocytes with hypertrophy induced by 10 μmol/L phenylephrine. Gain and loss of Rad function in cardiomyocytes significantly inhibited and increased phenylephrine-induced hypertrophy, respectively. In addition, activation of calcium-calmodulin–dependent kinase II (CaMKII), a strong inducer of cardiac hypertrophy, was significantly inhibited by Rad overexpression. Conversely, downregulation of CaMKIIδ by RNA interference technology attenuated the phenylephrine-induced hypertrophic response in cardiomyocytes in which Rad was also knocked down. To further elucidate the potential role of Rad in vivo, we generated Rad-deficient mice and demonstrated that they were more susceptible to cardiac hypertrophy associated with increased CaMKII phosphorylation than wild-type littermate controls. Conclusions The present data document for the first time that Rad is a novel mediator that inhibits cardiac hypertrophy through the CaMKII pathway. The present study will have significant implications for understanding the mechanisms of cardiac hypertrophy and setting the basis for the development of new strategies for treatment of cardiac hypertrophy. PMID:18056528

  20. RadNet Databases and Reports

    EPA Pesticide Factsheets

    EPA’s RadNet data are available for viewing in a searchable database or as PDF reports. Historical and current RadNet monitoring data are used to estimate long-term trends in environmental radiation levels.

  1. Lignin Hydrogenolysis: Improving Lignin Disassembly through Formaldehyde Stabilization

    PubMed Central

    2017-01-01

    Abstract Lignocellulosic biomass is available in large quantities and constitutes an attractive feedstock for the sustainable production of bulk and fine chemicals. Although methods have been established for the conversion of its cellulosic fractions, valorization of lignin has proven to be challenging. The difficulty in disassembling lignin originates from its heterogeneous structure and its propensity to undergo skeletal rearrangements and condensation reactions during biorefinery fractionation or biomass pretreatment processes. A strategy for hindering the generation of these resistive interunit linkages during biomass pretreatment has now been devised using formaldehyde as a stabilizing agent. The developed method when combined with Ru/C‐catalyzed hydrogenolysis allows for efficient disassembly of all three biomass fractions: (cellulose, hemicellulose, and lignin) and suggests that lignin upgrading can be integrated into prevailing biorefinery schemes. PMID:28394095

  2. Long repeating (TTAGGG)n single stranded DNA self-condenses into compact beaded filaments stabilized by G-quadruplex formation.

    PubMed

    Kar, Anirban; Jones, Nathan; Arat, N Özlem; Fishel, Richard; Griffith, Jack

    2018-04-19

    Conformations adopted by long stretches of single stranded DNA (ssDNA) are of central interest in understanding the architecture of replication forks, R loops, and other structures generated during DNA metabolism in vivo. This is particularly so if the ssDNA consists of short nucleotide repeats. Such studies have been hampered by the lack of defined substrates greater than ~150 nt, and the absence of high-resolution biophysical approaches. Here we describe the generation of very long ssDNA consisting of the mammalian telomeric repeat (5'-TTAGGG-3')n as well as the interrogation of its structure by electron microscopy (EM) and single molecule magnetic tweezers (smMT). This repeat is of particular interest as it contains a run of 3 contiguous guanine residues capable of forming G quartets as ssDNA. Fluorescent-dye exclusion assays confirmed that this G-strand ssDNA forms ubiquitous G-quadruplex folds. EM revealed thick bead-like filaments that condensed the DNA ~12 fold. The bead-like structures were 5 nm and 8 nm in diameter and linked by thin filaments. The G-strand ssDNA displayed initial stability to smMT force extension that ultimately released in steps that were multiples ~28 nm at forces between 6-12 pN; well below the >20 pN required to unravel G-quadruplexes. Most smMT steps were consistent with the disruption of the beads seen by EM. Binding by RAD51 distinctively altered the force extension properties of the G-strand ssDNA, suggesting a stochastic G-quadruplex-dependent condensation model that is discussed. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  3. The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis

    PubMed Central

    Nadarajan, Saravanapriah; Mohideen, Firaz; Tzur, Yonatan B; Ferrandiz, Nuria; Crawley, Oliver; Montoya, Alex; Faull, Peter; Snijders, Ambrosius P; Cutillas, Pedro R; Jambhekar, Ashwini; Blower, Michael D; Martinez-Perez, Enrique; Harper, J Wade; Colaiacovo, Monica P

    2016-01-01

    Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.12039.001 PMID:26920220

  4. Comparative Proteomics Reveals Timely Transport into Cilia of Regulators or Effectors as a Mechanism Underlying Ciliary Disassembly.

    PubMed

    Wang, Limei; Gu, Lixiao; Meng, Dan; Wu, Qiong; Deng, Haiteng; Pan, Junmin

    2017-07-07

    Primary cilia are assembled and disassembled during cell cycle progression. During ciliary disassembly, ciliary axonemal microtubules (MTs) are depolymerized accompanied by extensive posttranslational protein modifications of ciliary proteins including protein phosphorylation, methylation, and ubiquitination. These events are hypothesized to involve transport of effectors or regulators into cilia at the time of ciliary disassembly from the cell body. To prove this hypothesis and identify new proteins involved in ciliary disassembly, we analyzed disassembling flagella in Chlamydomonas using comparative proteomics with TMT labeling. Ninety-one proteins were found to increase more than 1.4-fold in four replicates. The proteins of the IFT machinery not only increase but also exhibit stoichiometric changes. The other proteins that increase include signaling molecules, chaperones, and proteins involved in microtubule dynamics or stability. In particular, we have identified a ciliopathy protein C21orf2, the AAA-ATPase CDC48, that is involved in segregating polypeptides from large assemblies or cellular structures, FAP203 and FAP236, which are homologous to stabilizers of axonemal microtubules. Our data demonstrate that ciliary transport of effectors or regulators is one of the mechanisms underlying ciliary disassembly. Further characterization of the proteins identified will provide new insights into our understanding of ciliary disassembly and likely ciliopathy.

  5. The Chloroplast Division Protein ARC6 Acts to Inhibit Disassembly of GDP-bound FtsZ2.

    PubMed

    Sung, Min Woo; Shaik, Rahamthulla; TerBush, Allan D; Osteryoung, Katherine W; Vitha, Stanislav; Holzenburg, Andreas

    2018-05-16

    Chloroplasts host photosynthesis and fulfill other metabolic functions that are essential to plant life. They have to divide by binary fission to maintain their numbers throughout cycles of cell division. Chloroplast division is achieved by a complex ring-shaped division machinery located on both the inner (stromal) and the outer (cytosolic) side of the chloroplast envelope. The inner division ring (termed the Z ring) is formed by the assembly of tubulin-like FtsZ1 and FtsZ2 proteins. ARC6 is a key chloroplast division protein that interacts with the Z ring. ARC6 spans the inner envelope membrane, is known to stabilize or maintain the Z ring, and anchors the Z ring to the inner membrane through interaction with FtsZ2. The underlying mechanism of Z-ring stabilization is not well understood. Here, biochemical and structural characterization of ARC6 was conducted using light scattering, sedimentation, and light and transmission electron microscopy. The recombinant protein was purified as a dimer. The results indicated that a truncated form of ARC6 (tARC6), representing the stromal portion of ARC6, affects FtsZ2 assembly without forming higher-order structures, and exerts its effect via FtsZ2 dynamics. tARC6 prevented GDP-induced FtsZ2 disassembly and caused a significant net increase in FtsZ2 assembly when GDP was present. Single particle analysis and 3D reconstruction were performed to elucidate the structural basis of ARC6 activity. Together, the data reveal that a dimeric form of tARC6 binds to FtsZ2 filaments and does not increase FtsZ polymerization rates but rather inhibits GDP-associated FtsZ2 disassembly. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Differential gene expression and filamentation of Listeria monocytogenes 08-5923 exposed to sodium lactate and sodium diacetate.

    PubMed

    Liu, Xiaoji; Basu, Urmila; Miller, Petr; McMullen, Lynn M

    2017-05-01

    This study reports the gene expression and filamentation in Listeria monocytogenes 08-5923 following exposure to food preservatives sodium lactate (NaL) and sodium diacetate (SD). L. monocytogenes 08-5923 was challenged with a mixture of NaL/SD, NaL or sodium acetate at 37 °C in tryptic soy broth. In the initial study, L. monocytogenes 08-5923 was exposed to NaL/SD for 24 h. The transcriptome was investigated by RNA sequencing. A stress response network was discovered in L. monocytogenes 08-5923, which is mediated by genes encoding two-component systems (hisJ, lisK, OmpR family gene, resE) and RNA polymerase factors (sigC, sigH). NaL/SD resulted in the down-regulation of genes in glycolysis (pykA, eno, fbaA, pgm) and up-regulation of genes in DNA repair (radC), cell division (ftsE) and cell structure synthesis (flagella synthesis: flgK, fliF, fliD). Filamentation was monitored by flow cytometry. NaL/SD mixture resulted in filamentation in L. monocytogenes 08-5923. Longer exposure was required to induce filamentation in L. monocytogenes for SD (24 h) than for NaL (8 h) when cells were exposed to individual salt. The quantitative real time PCR analysis revealed the down-regulation of ftsE in filamented cells of Listeria exposed to NaL or sodium acetate. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae.

    PubMed

    Saparbaev, M; Prakash, L; Prakash, S

    1996-03-01

    The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3 delta mutation has an effect on recombination similar to that of the rad1 delta and rad10 delta mutations. The msh2 delta mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process.

  8. Preliminary topical report on comparison reactor disassembly calculations

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

    McLaughlin, T.P.

    1975-11-01

    Preliminary results of comparison disassembly calculations for a representative LMFBR model (2100-l voided core) and arbitrary accident conditions are described. The analytical methods employed were the computer programs: FX2- POOL, PAD, and VENUS-II. The calculated fission energy depositions are in good agreement, as are measures of the destructive potential of the excursions, kinetic energy, and work. However, in some cases the resulting fuel temperatures are substantially divergent. Differences in the fission energy deposition appear to be attributable to residual inconsistencies in specifying the comparison cases. In contrast, temperature discrepancies probably stem from basic differences in the energy partition models inherentmore » in the codes. Although explanations of the discrepancies are being pursued, the preliminary results indicate that all three computational methods provide a consistent, global characterization of the contrived disassembly accident. (auth)« less

  9. Rad-deletion Phenocopies Tonic Sympathetic Stimulation of the Heart.

    PubMed

    Levitan, Bryana M; Manning, Janet R; Withers, Catherine N; Smith, Jeffrey D; Shaw, Robin M; Andres, Douglas A; Sorrell, Vincent L; Satin, Jonathan

    2016-12-01

    Sympathetic stimulation modulates L-type calcium channel (LTCC) gating to contribute to increased systolic heart function. Rad is a monomeric G-protein that interacts with LTCC. Genetic deletion of Rad (Rad -/- ) renders LTCC in a sympathomimetic state. The study goal was to use a clinically inspired pharmacological stress echocardiography test, including analysis of global strain, to determine whether Rad -/- confers tonic positive inotropic heart function. Sarcomere dynamics and strain showed partial parallel isoproterenol (ISO) responsiveness for wild-type (WT) and for Rad -/- . Rad -/- basal inotropy was elevated compared to WT but was less responsiveness to ISO. Rad protein levels were lower in human patients with end-stage non-ischemic heart failure. These results show that Rad reduction provides a stable inotropic response rooted in sarcomere level function. Thus, reduced Rad levels in heart failure patients may be a compensatory response to need for increased output in the setting of HF. Rad deletion suggests a future therapeutic direction for inotropic support.

  10. Direct measurement of conformational strain energy in protofilaments curling outward from disassembling microtubule tips.

    PubMed

    Driver, Jonathan W; Geyer, Elisabeth A; Bailey, Megan E; Rice, Luke M; Asbury, Charles L

    2017-06-19

    Disassembling microtubules can generate movement independently of motor enzymes, especially at kinetochores where they drive chromosome motility. A popular explanation is the 'conformational wave' model, in which protofilaments pull on the kinetochore as they curl outward from a disassembling tip. But whether protofilaments can work efficiently via this spring-like mechanism has been unclear. By modifying a previous assay to use recombinant tubulin and feedback-controlled laser trapping, we directly demonstrate the spring-like elasticity of curling protofilaments. Measuring their mechanical work output suggests they carry ~25% of the energy of GTP hydrolysis as bending strain, enabling them to drive movement with efficiency similar to conventional motors. Surprisingly, a β-tubulin mutant that dramatically slows disassembly has no effect on work output, indicating an uncoupling of disassembly speed from protofilament strain. These results show the wave mechanism can make a major contribution to kinetochore motility and establish a direct approach for measuring tubulin mechano-chemistry.

  11. Design and analysis of sustainable computer mouse using design for disassembly methodology

    NASA Astrophysics Data System (ADS)

    Roni Sahroni, Taufik; Fitri Sukarman, Ahmad; Agung Mahardini, Karunia

    2017-12-01

    This paper presents the design and analysis of computer mouse using Design for Disassembly methodology. Basically, the existing computer mouse model consist a number of unnecessary part that cause the assembly and disassembly time in production. The objective of this project is to design a new computer mouse based on Design for Disassembly (DFD) methodology. The main methodology of this paper was proposed from sketch generation, concept selection, and concept scoring. Based on the design screening, design concept B was selected for further analysis. New design of computer mouse is proposed using fastening system. Furthermore, three materials of ABS, Polycarbonate, and PE high density were prepared to determine the environmental impact category. Sustainable analysis was conducted using software SolidWorks. As a result, PE High Density gives the lowers amount in the environmental category with great maximum stress value.

  12. Force maintenance and myosin filament assembly regulated by Rho-kinase in airway smooth muscle.

    PubMed

    Lan, Bo; Deng, Linhong; Donovan, Graham M; Chin, Leslie Y M; Syyong, Harley T; Wang, Lu; Zhang, Jenny; Pascoe, Christopher D; Norris, Brandon A; Liu, Jeffrey C-Y; Swyngedouw, Nicholas E; Banaem, Saleha M; Paré, Peter D; Seow, Chun Y

    2015-01-01

    Smooth muscle contraction can be divided into two phases: the initial contraction determines the amount of developed force and the second phase determines how well the force is maintained. The initial phase is primarily due to activation of actomyosin interaction and is relatively well understood, whereas the second phase remains poorly understood. Force maintenance in the sustained phase can be disrupted by strains applied to the muscle; the strain causes actomyosin cross-bridges to detach and also the cytoskeletal structure to disassemble in a process known as fluidization, for which the underlying mechanism is largely unknown. In the present study we investigated the ability of airway smooth muscle to maintain force after the initial phase of contraction. Specifically, we examined the roles of Rho-kinase and protein kinase C (PKC) in force maintenance. We found that for the same degree of initial force inhibition, Rho-kinase substantially reduced the muscle's ability to sustain force under static conditions, whereas inhibition of PKC had a minimal effect on sustaining force. Under oscillatory strain, Rho-kinase inhibition caused further decline in force, but again, PKC inhibition had a minimal effect. We also found that Rho-kinase inhibition led to a decrease in the myosin filament mass in the muscle cells, suggesting that one of the functions of Rho-kinase is to stabilize myosin filaments. The results also suggest that dissolution of myosin filaments may be one of the mechanisms underlying the phenomenon of fluidization. These findings can shed light on the mechanism underlying deep inspiration induced bronchodilation. Copyright © 2015 the American Physiological Society.

  13. Force maintenance and myosin filament assembly regulated by Rho-kinase in airway smooth muscle

    PubMed Central

    Lan, Bo; Deng, Linhong; Donovan, Graham M.; Chin, Leslie Y. M.; Syyong, Harley T.; Wang, Lu; Zhang, Jenny; Pascoe, Christopher D.; Norris, Brandon A.; Liu, Jeffrey C.-Y.; Swyngedouw, Nicholas E.; Banaem, Saleha M.; Paré, Peter D.

    2014-01-01

    Smooth muscle contraction can be divided into two phases: the initial contraction determines the amount of developed force and the second phase determines how well the force is maintained. The initial phase is primarily due to activation of actomyosin interaction and is relatively well understood, whereas the second phase remains poorly understood. Force maintenance in the sustained phase can be disrupted by strains applied to the muscle; the strain causes actomyosin cross-bridges to detach and also the cytoskeletal structure to disassemble in a process known as fluidization, for which the underlying mechanism is largely unknown. In the present study we investigated the ability of airway smooth muscle to maintain force after the initial phase of contraction. Specifically, we examined the roles of Rho-kinase and protein kinase C (PKC) in force maintenance. We found that for the same degree of initial force inhibition, Rho-kinase substantially reduced the muscle's ability to sustain force under static conditions, whereas inhibition of PKC had a minimal effect on sustaining force. Under oscillatory strain, Rho-kinase inhibition caused further decline in force, but again, PKC inhibition had a minimal effect. We also found that Rho-kinase inhibition led to a decrease in the myosin filament mass in the muscle cells, suggesting that one of the functions of Rho-kinase is to stabilize myosin filaments. The results also suggest that dissolution of myosin filaments may be one of the mechanisms underlying the phenomenon of fluidization. These findings can shed light on the mechanism underlying deep inspiration induced bronchodilation. PMID:25305246

  14. T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures.

    PubMed

    Schaffter, Samuel W; Green, Leopold N; Schneider, Joanna; Subramanian, Hari K K; Schulman, Rebecca; Franco, Elisa

    2018-06-01

    The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases.

  15. PI-RADS v2: Current standing and future outlook.

    PubMed

    Smith, Clayton P; Türkbey, Barış

    2018-05-01

    The Prostate Imaging-Reporting and Data System (PI-RADS) was created in 2012 to establish standardization in prostate multiparametric magnetic resonance imaging (mpMRI) acquisition, interpretation, and reporting. In hopes of improving upon some of the PI-RADS v1 shortcomings, the PI-RADS Steering Committee released PI-RADS v2 in 2015. This paper reviews the accuracy, interobserver agreement, and clinical outcomes of PI-RADS v2 and comments on the limitations of the current literature. Overall, PI-RADS v2 shows improved sensitivity and similar specificity compared to PI-RADS v1. However, concerns exist regarding interobserver agreement and the heterogeneity of the study methodology.

  16. PI-RADS v2: Current standing and future outlook

    PubMed Central

    Smith, Clayton P.

    2018-01-01

    The Prostate Imaging-Reporting and Data System (PI-RADS) was created in 2012 to establish standardization in prostate multiparametric magnetic resonance imaging (mpMRI) acquisition, interpretation, and reporting. In hopes of improving upon some of the PI-RADS v1 shortcomings, the PI-RADS Steering Committee released PI-RADS v2 in 2015. This paper reviews the accuracy, interobserver agreement, and clinical outcomes of PI-RADS v2 and comments on the limitations of the current literature. Overall, PI-RADS v2 shows improved sensitivity and similar specificity compared to PI-RADS v1. However, concerns exist regarding interobserver agreement and the heterogeneity of the study methodology. PMID:29733790

  17. Construction of two whole genome radiation hybrid panels for dromedary (Camelus dromedarius): 5000RAD and 15000RAD.

    PubMed

    Perelman, Polina L; Pichler, Rudolf; Gaggl, Anna; Larkin, Denis M; Raudsepp, Terje; Alshanbari, Fahad; Holl, Heather M; Brooks, Samantha A; Burger, Pamela A; Periasamy, Kathiravan

    2018-01-31

    The availability of genomic resources including linkage information for camelids has been very limited. Here, we describe the construction of a set of two radiation hybrid (RH) panels (5000 RAD and 15000 RAD ) for the dromedary (Camelus dromedarius) as a permanent genetic resource for camel genome researchers worldwide. For the 5000 RAD panel, a total of 245 female camel-hamster radiation hybrid clones were collected, of which 186 were screened with 44 custom designed marker loci distributed throughout camel genome. The overall mean retention frequency (RF) of the final set of 93 hybrids was 47.7%. For the 15000 RAD panel, 238 male dromedary-hamster radiation hybrid clones were collected, of which 93 were tested using 44 PCR markers. The final set of 90 clones had a mean RF of 39.9%. This 15000 RAD panel is an important high-resolution complement to the main 5000 RAD panel and an indispensable tool for resolving complex genomic regions. This valuable genetic resource of dromedary RH panels is expected to be instrumental for constructing a high resolution camel genome map. Construction of the set of RH panels is essential step toward chromosome level reference quality genome assembly that is critical for advancing camelid genomics and the development of custom genomic tools.

  18. CALUTRON ASSEMBLING AND DISASSEMBLING MEANS

    DOEpatents

    Andrews, R.E.; Thornton, J.

    1959-01-27

    This patent relates to the assembling and disassembling of a calutron and, more specifically describes a calutron having the ion separating mechanism carried by a fuce plate removably secured to the tank. When it is desired to withdraw the ion separating mechanism from the tank, a motor is energized and a carriage attached through a bracket to the fuce plate is driven along a track. The face plate moves out from the tank in substantially a linear direction, preventing injury to the ion separating mechanism.

  19. The assembly and disassembly of ecological networks.

    PubMed

    Bascompte, Jordi; Stouffer, Daniel B

    2009-06-27

    Global change has created a severe biodiversity crisis. Species are driven extinct at an increasing rate, and this has the potential to cause further coextinction cascades. The rate and shape of these coextinction cascades depend very much on the structure of the networks of interactions across species. Understanding network structure and how it relates to network disassembly, therefore, is a priority for system-level conservation biology. This process of network collapse may indeed be related to the process of network build-up, although very little is known about both processes and even less about their relationship. Here we review recent work that provides some preliminary answers to these questions. First, we focus on network assembly by emphasizing temporal processes at the species level, as well as the structural building blocks of complex ecological networks. Second, we focus on network disassembly as a consequence of species extinctions or habitat loss. We conclude by emphasizing some general rules of thumb that can help in building a comprehensive framework to understand the responses of ecological networks to global change.

  20. Diagnosis of femtosecond plasma filament by channeling microwaves along the filament

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

    Alshershby, Mostafa; Ren, Yu; Qin, Jiang

    2013-05-20

    We introduce a simple, fast, and non-intrusive experimental method to obtain the basic parameters of femtosecond laser-generated plasma filament. The method is based on the channeling of microwaves along both a plasma filament and a well-defined conducting wire. By comparing the detected microwaves that propagate along the plasma filament and a copper wire with known conductivity and spatial dimension, the basic parameters of the plasma filament can be easily obtained. As a result of the possibility of channeling microwave radiation along the plasma filament, we were then able to obtain the plasma density distribution along the filament length.

  1. Disassembly properties and material characterisation of household small waste electric and electronic equipment.

    PubMed

    Bovea, María D; Pérez-Belis, Victoria; Ibáñez-Forés, Valeria; Quemades-Beltrán, Pilar

    2016-07-01

    This paper is focused on characterising small waste electric and electronic equipment, specifically small household appliances, from two different points of views: disassembly properties and material identification. The sample for this characterisation was obtained from a selective collection campaign organised in Castellón de la Plana (Spain). A total amount of 833.7kg (749 units) of small waste electric and electronic equipment was collected, of which 23.3% by weight and 22.4% by units belonged to the subcategory household equipment. This subcategory, composed of appliances such as vacuum cleaners, toasters, sandwich makers, hand blenders, juicers, coffee makers, hairdryers, scales, irons and heaters, was first disassembled in order to analyse different aspects of the disassembly process for each equipment type: type of joints, ease of identification of materials, ease of access to joints for extracting components, ease of separation of components from the whole, uniformity of tools needed for the disassembly process and possibility of reassembly after disassembly. Results show that the most common joints used in these equipment types are snap-fits and screws, although some permanent joints have also been identified. Next, the material composition of each component of each appliance belonging to each equipment type was identified visually and with additional mechanical trials and testing. It can be observed that plastic and electric/electronic components are present in all the equipment types analysed and are also the material fractions that appear with higher percentages in the material composition: 41.1wt% and 39.1wt% for the plastic fraction and electric/electronic components, respectively. The most common plastics are: polypropylene (PP), acrylonitrile butadiene styrene (ABS) and polycarbonate (PC), while the most common electric/electronic components are: cable, plug and printed circuit boards. Results also show that disassembly properties and material

  2. RadVel: The Radial Velocity Modeling Toolkit

    NASA Astrophysics Data System (ADS)

    Fulton, Benjamin J.; Petigura, Erik A.; Blunt, Sarah; Sinukoff, Evan

    2018-04-01

    RadVel is an open-source Python package for modeling Keplerian orbits in radial velocity (RV) timeseries. RadVel provides a convenient framework to fit RVs using maximum a posteriori optimization and to compute robust confidence intervals by sampling the posterior probability density via Markov Chain Monte Carlo (MCMC). RadVel allows users to float or fix parameters, impose priors, and perform Bayesian model comparison. We have implemented real-time MCMC convergence tests to ensure adequate sampling of the posterior. RadVel can output a number of publication-quality plots and tables. Users may interface with RadVel through a convenient command-line interface or directly from Python. The code is object-oriented and thus naturally extensible. We encourage contributions from the community. Documentation is available at http://radvel.readthedocs.io.

  3. Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae

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

    Saparbaev, M.; Prakash, L.; Prakash, S.

    1996-03-01

    The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3-A mutation has an effect on recombination similar to that of the rad1{Delta} and rad10{Delta} mutations. The msh2{Delta} mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integrationmore » of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAM-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process. 59 refs., 2 figs., 7 tabs.« less

  4. RadNet Air Quality (Fixed Station) Data

    EPA Pesticide Factsheets

    RadNet is a national network of monitoring stations that regularly collect air for analysis of radioactivity. The RadNet network, which has stations in each State, has been used to track environmental releases of radioactivity from nuclear weapons tests and nuclear accidents. RadNet also documents the status and trends of environmental radioactivity

  5. Direct measurement of conformational strain energy in protofilaments curling outward from disassembling microtubule tips

    PubMed Central

    Driver, Jonathan W; Geyer, Elisabeth A; Bailey, Megan E; Rice, Luke M; Asbury, Charles L

    2017-01-01

    Disassembling microtubules can generate movement independently of motor enzymes, especially at kinetochores where they drive chromosome motility. A popular explanation is the ‘conformational wave’ model, in which protofilaments pull on the kinetochore as they curl outward from a disassembling tip. But whether protofilaments can work efficiently via this spring-like mechanism has been unclear. By modifying a previous assay to use recombinant tubulin and feedback-controlled laser trapping, we directly demonstrate the spring-like elasticity of curling protofilaments. Measuring their mechanical work output suggests they carry ~25% of the energy of GTP hydrolysis as bending strain, enabling them to drive movement with efficiency similar to conventional motors. Surprisingly, a β-tubulin mutant that dramatically slows disassembly has no effect on work output, indicating an uncoupling of disassembly speed from protofilament strain. These results show the wave mechanism can make a major contribution to kinetochore motility and establish a direct approach for measuring tubulin mechano-chemistry. DOI: http://dx.doi.org/10.7554/eLife.28433.001 PMID:28628007

  6. Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens

    PubMed Central

    Suchan, Tomasz; Pitteloud, Camille; Gerasimova, Nadezhda S.; Kostikova, Anna; Schmid, Sarah; Arrigo, Nils; Pajkovic, Mila; Ronikier, Michał; Alvarez, Nadir

    2016-01-01

    In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes

  7. RadMap

    EPA Pesticide Factsheets

    RadMap is an interactive desktop tool featuring a nationwide geographic information systems (GIS) map of long-term radiation monitoring locations across the United States with access to key information about the monitor and the area surrounding it.

  8. Regulated assembly and disassembly of the yeast telomerase quaternary complex

    PubMed Central

    Tucey, Timothy M.

    2014-01-01

    The enzyme telomerase, which elongates chromosome termini, is a critical factor in determining long-term cellular proliferation and tissue renewal. Hence, even small differences in telomerase levels can have substantial consequences for human health. In budding yeast, telomerase consists of the catalytic Est2 protein and two regulatory subunits (Est1 and Est3) in association with the TLC1 RNA, with each of the four subunits essential for in vivo telomerase function. We show here that a hierarchy of assembly and disassembly results in limiting amounts of the quaternary complex late in the cell cycle, following completion of DNA replication. The assembly pathway, which is driven by interaction of the Est3 telomerase subunit with a previously formed Est1–TLC1–Est2 preassembly complex, is highly regulated, involving Est3-binding sites on both Est2 and Est1 as well as an interface on Est3 itself that functions as a toggle switch. Telomerase subsequently disassembles by a mechanistically distinct pathway due to dissociation of the catalytic subunit from the complex in every cell cycle. The balance between the assembly and disassembly pathways, which dictate the levels of the active holoenzyme in the cell, reveals a novel mechanism by which telomerase (and hence telomere homeostasis) is regulated. PMID:25240060

  9. Three New Genetic Loci (R1210C in CFH, Variants in COL8A1 and RAD51B) Are Independently Related to Progression to Advanced Macular Degeneration

    PubMed Central

    Seddon, Johanna M.; Reynolds, Robyn; Yu, Yi; Rosner, Bernard

    2014-01-01

    Objectives To assess the independent impact of new genetic variants on conversion to advanced stages of AMD, controlling for established risk factors, and to determine the contribution of genes in predictive models. Methods In this prospective longitudinal study of 2765 individuals, 777 subjects progressed to neovascular disease (NV) or geographic atrophy (GA) in either eye over 12 years. Recently reported genetic loci were assessed for their independent effects on incident advanced AMD after controlling for 6 established loci in 5 genes, and demographic, behavioral, and macular characteristics. New variants which remained significantly related to progression were then added to a final multivariate model to assess their independent effects. The contribution of genes to risk models was assessed using reclassification tables by determining risk within cross-classified quintiles for alternative models. Results Three new genetic variants were significantly related to progression: rare variant R1210C in CFH (hazard ratio (HR) 2.5, 95% confidence interval [CI] 1.2–5.3, P = 0.01), and common variants in genes COL8A1 (HR 2.0, 95% CI 1.1–3.5, P = 0.02) and RAD51B (HR 0.8, 95% CI 0.60–0.97, P = 0.03). The area under the curve statistic (AUC) was significantly higher for the 9 gene model (.884) vs the 0 gene model (.873), P = .01. AUC’s for the 9 vs 6 gene models were not significantly different, but reclassification analyses indicated significant added information for more genes, with adjusted odds ratios (OR) for progression within 5 years per one quintile increase in risk score of 2.7, P<0.001 for the 9 vs 6 loci model, and OR 3.5, P<0.001 for the 9 vs. 0 gene model. Similar results were seen for NV and GA. Conclusions Rare variant CFH R1210C and common variants in COL8A1 and RAD51B plus six genes in previous models contribute additional predictive information for advanced AMD beyond macular and behavioral phenotypes. PMID:24498017

  10. T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures

    PubMed Central

    Schaffter, Samuel W; Green, Leopold N; Schneider, Joanna; Subramanian, Hari K K; Schulman, Rebecca

    2018-01-01

    Abstract The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases. PMID:29718412

  11. Deactivation of the P, C, and R Reactor Disassembly Basins at the SRS

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

    Pickett, J.B.

    The Facilities Disposition Division (FDD) at the Savannah River Site is engaged in planning the deactivation/closure of three of the site's five reactor disassembly basins. Activities are currently underway at 105-R Disassembly Basin and will continue with the 105-P and 105-C disassembly basins. The basins still contain the cooling and shielding water that was present when operations ceased. Low concentrations of radionuclides are present, with tritium, Cs-137, and Sr-90 being the major contributors. Although there is no evidence that any of the basins have leaked, the 50-year-old facilities will eventually contaminate the surrounding groundwaters. The FDD is pursuing a pro-activemore » solution to close the basins in-place and prevent a release to the groundwater. In-situ ion-exchange is currently underway at the R-Reactor Disassembly Basin to reduce the Cs and Sr concentrations to levels that would allow release of the treated water to previously used on-site cooling ponds. A NEPA Environm ental Assessment (EA) is being prepared to propose the preferred closure alternative for each of the three basins. The EA will be the primary mechanism to inform the public and gain stakeholder and regulatory approval.« less

  12. Deactivation of the P, C, and R Reactor Disassembly Basins at the SRS

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

    Pickett, J.B.

    The Facilities Disposition Division (FDD) at the Savannah River Site is engaged in planning the deactivation/closure of three of the site's five reactor disassembly basins. Activities are currently underway at 105-R Disassembly Basin and will continue with the 105-P and 105-C disassembly basins. The basins still contain the cooling and shielding water that was present when operations ceased. Low concentrations of radionuclides are present, with tritium, Cs-137, and Sr-90 being the major contributors. Although there is no evidence that any of the basins have leaked, the 50-year-old facilities will eventually contaminate the surrounding groundwaters. The FDD is pursuing a pro-activemore » solution to close the basins in-place and prevent a release to the groundwater. In-situ ion-exchange is currently underway at the R-Reactor Disassembly Basin to reduce the Cs and Sr concentrations to levels that would allow release of the treated water to previously used on-site cooling ponds. A NEPA Environmental Assessment (EA) is being prepared to propose the preferred closure alternative for each of the three basins. The EA will be the primary mechanism to inform the public and gain stakeholder and regulatory approval.« less

  13. Kinetics of Surface-Driven Self-Assembly and Fatigue-Induced Disassembly of a Virus-Based Nanocoating.

    PubMed

    Valbuena, Alejandro; Mateu, Mauricio G

    2017-02-28

    Self-assembling protein layers provide a "bottom-up" approach for precisely organizing functional elements at the nanoscale over a large solid surface area. The design of protein sheets with architecture and physical properties suitable for nanotechnological applications may be greatly facilitated by a thorough understanding of the principles that underlie their self-assembly and disassembly. In a previous study, the hexagonal lattice formed by the capsid protein (CA) of human immunodeficiency virus (HIV) was self-assembled as a monomolecular layer directly onto a solid substrate, and its mechanical properties and dynamics at equilibrium were analyzed by atomic force microscopy. Here, we use atomic force microscopy to analyze the kinetics of self-assembly of the planar CA lattice on a substrate and of its disassembly, either spontaneous or induced by materials fatigue. Both self-assembly and disassembly of the CA layer are cooperative reactions that proceed until a phase equilibrium is reached. Self-assembly requires a critical protein concentration and is initiated by formation of nucleation points on the substrate, followed by lattice growth and eventual merging of CA patches into a continuous monolayer. Disassembly of the CA layer showed hysteresis and appears to proceed only after large enough defects (nucleation points) are formed in the lattice, whose number is largely increased by inducing materials fatigue that depends on mechanical load and its frequency. Implications of the kinetic results obtained for a better understanding of self-assembly and disassembly of the HIV capsid and protein-based two-dimensional nanomaterials and the design of anti-HIV drugs targeting (dis)assembly and biocompatible nanocoatings are discussed. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  14. RadNet Sampling and Analyses Schedules

    EPA Pesticide Factsheets

    RadNet air monitors operate continuously and samples of air, precipitation and drinking water and analyzed on a routine schedule. RadNet can send deployable monitors to any U.S. location in the case of a radiological emergency.

  15. Structured reporting platform improves CAD-RADS assessment.

    PubMed

    Szilveszter, Bálint; Kolossváry, Márton; Karády, Júlia; Jermendy, Ádám L; Károlyi, Mihály; Panajotu, Alexisz; Bagyura, Zsolt; Vecsey-Nagy, Milán; Cury, Ricardo C; Leipsic, Jonathon A; Merkely, Béla; Maurovich-Horvat, Pál

    2017-11-01

    Structured reporting in cardiac imaging is strongly encouraged to improve quality through consistency. The Coronary Artery Disease - Reporting and Data System (CAD-RADS) was recently introduced to facilitate interdisciplinary communication of coronary CT angiography (CTA) results. We aimed to assess the agreement between manual and automated CAD-RADS classification using a structured reporting platform. Five readers prospectively interpreted 500 coronary CT angiographies using a structured reporting platform that automatically calculates the CAD-RADS score based on stenosis and plaque parameters manually entered by the reader. In addition, all readers manually assessed CAD-RADS blinded to the automatically derived results, which was used as the reference standard. We evaluated factors influencing reader performance including CAD-RADS training, clinical load, time of the day and level of expertise. Total agreement between manual and automated classification was 80.2%. Agreement in stenosis categories was 86.7%, whereas the agreement in modifiers was 95.8% for "N", 96.8% for "S", 95.6% for "V" and 99.4% for "G". Agreement for V improved after CAD-RADS training (p = 0.047). Time of the day and clinical load did not influence reader performance (p > 0.05 both). Less experienced readers had a higher total agreement as compared to more experienced readers (87.0% vs 78.0%, respectively; p = 0.011). Even though automated CAD-RADS classification uses data filled in by the readers, it outperforms manual classification by preventing human errors. Structured reporting platforms with automated calculation of the CAD-RADS score might improve data quality and support standardization of clinical decision making. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Metrics and experimental data for assessing unbalanced disassembly lines.

    DOT National Transportation Integrated Search

    2011-01-01

    Disassembly lines are inherently multi-criteria, with balance having the possibility of : being one of the lower priorities. This is due to the fact that other criteria for example, : removing valuable or hazardous materials early on in the proce...

  17. RAD18 mediates resistance to ionizing radiation in human glioma cells

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

    Xie, Chen; Wang, Hongwei; Cheng, Hongbin

    Highlights: • RAD18 is an important mediator of the IR-induced resistance in glioma cell lines. • RAD18 overexpression confers resistance to IR-mediated apoptosis. • The elevated expression of RAD18 is associated with recurrent GBM who underwent IR therapy. - Abstract: Radioresistance remains a major challenge in the treatment of glioblastoma multiforme (GBM). RAD18 a central regulator of translesion DNA synthesis (TLS), has been shown to play an important role in regulating genomic stability and DNA damage response. In the present study, we investigate the relationship between RAD18 and resistance to ionizing radiation (IR) and examined the expression levels of RAD18more » in primary and recurrent GBM specimens. Our results showed that RAD18 is an important mediator of the IR-induced resistance in GBM. The expression level of RAD18 in glioma cells correlates with their resistance to IR. Ectopic expression of RAD18 in RAD18-low A172 glioma cells confers significant resistance to IR treatment. Conversely, depletion of endogenous RAD18 in RAD18-high glioma cells sensitized these cells to IR treatment. Moreover, RAD18 overexpression confers resistance to IR-mediated apoptosis in RAD18-low A172 glioma cells, whereas cells deficient in RAD18 exhibit increased apoptosis induced by IR. Furthermore, knockdown of RAD18 in RAD18-high glioma cells disrupts HR-mediated repair, resulting in increased accumulation of DSB. In addition, clinical data indicated that RAD18 was significantly higher in recurrent GBM samples that were exposed to IR compared with the corresponding primary GBM samples. Collectively, our findings reveal that RAD18 may serve as a key mediator of the IR response and may function as a potential target for circumventing IR resistance in human GBM.« less

  18. 29 CFR 1926.1407 - Power line safety (up to 350 kV)-assembly and disassembly.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 8 2014-07-01 2014-07-01 false Power line safety (up to 350 kV)-assembly and disassembly. 1926.1407 Section 1926.1407 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND... Cranes and Derricks in Construction § 1926.1407 Power line safety (up to 350 kV)—assembly and disassembly...

  19. Associations of common variants at 1p11.2 and 14q24.1 (RAD51L1) with breast cancer risk and heterogeneity by tumor subtype: findings from the Breast Cancer Association Consortium.

    PubMed

    Figueroa, Jonine D; Garcia-Closas, Montserrat; Humphreys, Manjeet; Platte, Radka; Hopper, John L; Southey, Melissa C; Apicella, Carmel; Hammet, Fleur; Schmidt, Marjanka K; Broeks, Annegien; Tollenaar, Rob A E M; Van't Veer, Laura J; Fasching, Peter A; Beckmann, Matthias W; Ekici, Arif B; Strick, Reiner; Peto, Julian; dos Santos Silva, Isabel; Fletcher, Olivia; Johnson, Nichola; Sawyer, Elinor; Tomlinson, Ian; Kerin, Michael; Burwinkel, Barbara; Marme, Federik; Schneeweiss, Andreas; Sohn, Christof; Bojesen, Stig; Flyger, Henrik; Nordestgaard, Børge G; Benítez, Javier; Milne, Roger L; Ignacio Arias, Jose; Zamora, M Pilar; Brenner, Hermann; Müller, Heiko; Arndt, Volker; Rahman, Nazneen; Turnbull, Clare; Seal, Sheila; Renwick, Anthony; Brauch, Hiltrud; Justenhoven, Christina; Brüning, Thomas; Chang-Claude, Jenny; Hein, Rebecca; Wang-Gohrke, Shan; Dörk, Thilo; Schürmann, Peter; Bremer, Michael; Hillemanns, Peter; Nevanlinna, Heli; Heikkinen, Tuomas; Aittomäki, Kristiina; Blomqvist, Carl; Bogdanova, Natalia; Antonenkova, Natalia; Rogov, Yuri I; Karstens, Johann Hinrich; Bermisheva, Marina; Prokofieva, Darya; Gantcev, Shamil Hanafievich; Khusnutdinova, Elza; Lindblom, Annika; Margolin, Sara; Chenevix-Trench, Georgia; Beesley, Jonathan; Chen, Xiaoqing; Mannermaa, Arto; Kosma, Veli-Matti; Soini, Ylermi; Kataja, Vesa; Lambrechts, Diether; Yesilyurt, Betül T; Chrisiaens, Marie-Rose; Peeters, Stephanie; Radice, Paolo; Peterlongo, Paolo; Manoukian, Siranoush; Barile, Monica; Couch, Fergus; Lee, Adam M; Diasio, Robert; Wang, Xianshu; Giles, Graham G; Severi, Gianluca; Baglietto, Laura; Maclean, Catriona; Offit, Ken; Robson, Mark; Joseph, Vijai; Gaudet, Mia; John, Esther M; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Grip, Mervi; Andrulis, Irene; Knight, Julia A; Mulligan, Anna Marie; O'Malley, Frances P; Brinton, Louise A; Sherman, Mark E; Lissowska, Jolanta; Chanock, Stephen J; Hooning, Maartje; Martens, John W M; van den Ouweland, Ans M W; Collée, J Margriet; Hall, Per; Czene, Kamila; Cox, Angela; Brock, Ian W; Reed, Malcolm W R; Cross, Simon S; Pharoah, Paul; Dunning, Alison M; Kang, Daehee; Yoo, Keun-Young; Noh, Dong-Young; Ahn, Sei-Hyun; Jakubowska, Anna; Lubinski, Jan; Jaworska, Katarzyna; Durda, Katarzyna; Sangrajrang, Suleeporn; Gaborieau, Valerie; Brennan, Paul; McKay, James; Shen, Chen-Yang; Ding, Shian-ling; Hsu, Huan-Ming; Yu, Jyh-Cherng; Anton-Culver, Hoda; Ziogas, Argyrios; Ashworth, Alan; Swerdlow, Anthony; Jones, Michael; Orr, Nick; Trentham-Dietz, Amy; Egan, Kathleen; Newcomb, Polly; Titus-Ernstoff, Linda; Easton, Doug; Spurdle, Amanda B

    2011-12-01

    A genome-wide association study (GWAS) identified single-nucleotide polymorphisms (SNPs) at 1p11.2 and 14q24.1 (RAD51L1) as breast cancer susceptibility loci. The initial GWAS suggested stronger effects for both loci for estrogen receptor (ER)-positive tumors. Using data from the Breast Cancer Association Consortium (BCAC), we sought to determine whether risks differ by ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), grade, node status, tumor size, and ductal or lobular morphology. We genotyped rs11249433 at 1p.11.2, and two highly correlated SNPs rs999737 and rs10483813 (r(2)= 0.98) at 14q24.1 (RAD51L1), for up to 46 036 invasive breast cancer cases and 46 930 controls from 39 studies. Analyses by tumor characteristics focused on subjects reporting to be white women of European ancestry and were based on 25 458 cases, of which 87% had ER data. The SNP at 1p11.2 showed significantly stronger associations with ER-positive tumors [per-allele odds ratio (OR) for ER-positive tumors was 1.13, 95% CI = 1.10-1.16 and, for ER-negative tumors, OR was 1.03, 95% CI = 0.98-1.07, case-only P-heterogeneity = 7.6 × 10(-5)]. The association with ER-positive tumors was stronger for tumors of lower grade (case-only P= 6.7 × 10(-3)) and lobular histology (case-only P= 0.01). SNPs at 14q24.1 were associated with risk for most tumor subtypes evaluated, including triple-negative breast cancers, which has not been described previously. Our results underscore the need for large pooling efforts with tumor pathology data to help refine risk estimates for SNP associations with susceptibility to different subtypes of breast cancer.

  20. Associations of common variants at 1p11.2 and 14q24.1 (RAD51L1) with breast cancer risk and heterogeneity by tumor subtype: findings from the Breast Cancer Association Consortium†

    PubMed Central

    Figueroa, Jonine D.; Garcia-Closas, Montserrat; Humphreys, Manjeet; Platte, Radka; Hopper, John L.; Southey, Melissa C.; Apicella, Carmel; Hammet, Fleur; Schmidt, Marjanka K.; Broeks, Annegien; Tollenaar, Rob A.E.M.; Van't Veer, Laura J.; Fasching, Peter A.; Beckmann, Matthias W.; Ekici, Arif B.; Strick, Reiner; Peto, Julian; dos Santos Silva, Isabel; Fletcher, Olivia; Johnson, Nichola; Sawyer, Elinor; Tomlinson, Ian; Kerin, Michael; Burwinkel, Barbara; Marme, Federik; Schneeweiss, Andreas; Sohn, Christof; Bojesen, Stig; Flyger, Henrik; Nordestgaard, Børge G.; Benítez, Javier; Milne, Roger L.; Ignacio Arias, Jose; Zamora, M. Pilar; Brenner, Hermann; Müller, Heiko; Arndt, Volker; Rahman, Nazneen; Turnbull, Clare; Seal, Sheila; Renwick, Anthony; Brauch, Hiltrud; Justenhoven, Christina; Brüning, Thomas; Chang-Claude, Jenny; Hein, Rebecca; Wang-Gohrke, Shan; Dörk, Thilo; Schürmann, Peter; Bremer, Michael; Hillemanns, Peter; Nevanlinna, Heli; Heikkinen, Tuomas; Aittomäki, Kristiina; Blomqvist, Carl; Bogdanova, Natalia; Antonenkova, Natalia; Rogov, Yuri I.; Karstens, Johann Hinrich; Bermisheva, Marina; Prokofieva, Darya; Hanafievich Gantcev, Shamil; Khusnutdinova, Elza; Lindblom, Annika; Margolin, Sara; Chenevix-Trench, Georgia; Beesley, Jonathan; Chen, Xiaoqing; Mannermaa, Arto; Kosma, Veli-Matti; Soini, Ylermi; Kataja, Vesa; Lambrechts, Diether; Yesilyurt, Betül T.; Chrisiaens, Marie-Rose; Peeters, Stephanie; Radice, Paolo; Peterlongo, Paolo; Manoukian, Siranoush; Barile, Monica; Couch, Fergus; Lee, Adam M.; Diasio, Robert; Wang, Xianshu; Giles, Graham G.; Severi, Gianluca; Baglietto, Laura; Maclean, Catriona; Offit, Ken; Robson, Mark; Joseph, Vijai; Gaudet, Mia; John, Esther M.; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Grip, Mervi; Andrulis, Irene; Knight, Julia A.; Marie Mulligan, Anna; O'Malley, Frances P.; Brinton, Louise A.; Sherman, Mark E.; Lissowska, Jolanta; Chanock, Stephen J.; Hooning, Maartje; Martens, John W.M.; van den Ouweland, Ans M.W.; Collée, J. Margriet; Hall, Per; Czene, Kamila; Cox, Angela; Brock, Ian W.; Reed, Malcolm W.R.; Cross, Simon S.; Pharoah, Paul; Dunning, Alison M.; Kang, Daehee; Yoo, Keun-Young; Noh, Dong-Young; Ahn, Sei-Hyun; Jakubowska, Anna; Lubinski, Jan; Jaworska, Katarzyna; Durda, Katarzyna; Sangrajrang, Suleeporn; Gaborieau, Valerie; Brennan, Paul; McKay, James; Shen, Chen-Yang; Ding, Shian-ling; Hsu, Huan-Ming; Yu, Jyh-Cherng; Anton-Culver, Hoda; Ziogas, Argyrios; Ashworth, Alan; Swerdlow, Anthony; Jones, Michael; Orr, Nick; Trentham-Dietz, Amy; Egan, Kathleen; Newcomb, Polly; Titus-Ernstoff, Linda; Easton, Doug; Spurdle, Amanda B.

    2011-01-01

    A genome-wide association study (GWAS) identified single-nucleotide polymorphisms (SNPs) at 1p11.2 and 14q24.1 (RAD51L1) as breast cancer susceptibility loci. The initial GWAS suggested stronger effects for both loci for estrogen receptor (ER)-positive tumors. Using data from the Breast Cancer Association Consortium (BCAC), we sought to determine whether risks differ by ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), grade, node status, tumor size, and ductal or lobular morphology. We genotyped rs11249433 at 1p.11.2, and two highly correlated SNPs rs999737 and rs10483813 (r2= 0.98) at 14q24.1 (RAD51L1), for up to 46 036 invasive breast cancer cases and 46 930 controls from 39 studies. Analyses by tumor characteristics focused on subjects reporting to be white women of European ancestry and were based on 25 458 cases, of which 87% had ER data. The SNP at 1p11.2 showed significantly stronger associations with ER-positive tumors [per-allele odds ratio (OR) for ER-positive tumors was 1.13, 95% CI = 1.10–1.16 and, for ER-negative tumors, OR was 1.03, 95% CI = 0.98–1.07, case-only P-heterogeneity = 7.6 × 10−5]. The association with ER-positive tumors was stronger for tumors of lower grade (case-only P= 6.7 × 10−3) and lobular histology (case-only P= 0.01). SNPs at 14q24.1 were associated with risk for most tumor subtypes evaluated, including triple-negative breast cancers, which has not been described previously. Our results underscore the need for large pooling efforts with tumor pathology data to help refine risk estimates for SNP associations with susceptibility to different subtypes of breast cancer. PMID:21852249

  1. Lymphoid irradiation in intractable rheumatoid arthritis. A double-blind, randomized study comparing 750-rad treatment with 2,000-rad treatment

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

    Hanly, J.G.; Hassan, J.; Moriarty, M.

    1986-01-01

    Twenty patients with intractable rheumatoid arthritis were treated with 750-rad or 2,000-rad lymphoid irradiation in a randomized double-blind comparative study. Over a 12-month followup period, there was a significant improvement in 4 of 7 and 6 of 7 standard parameters of disease activity following treatment with 750 rads and 2,000 rads, respectively. Transient, short-term toxicity was less frequent with the lower dose. In both groups, there was a sustained peripheral blood lymphopenia, a selective depletion of T helper (Leu-3a+) lymphocytes, and reduced in vitro mitogen responses. These changes did not occur, however, in synovial fluid. These results suggest that 750-radmore » lymphoid irradiation is as effective as, but less toxic than, that with 2,000 rads in the management of patients with intractable rheumatoid arthritis.« less

  2. Myosin-dependent remodeling of adherens junctions protects junctions from Snail-dependent disassembly

    PubMed Central

    Weng, Mo

    2016-01-01

    Although Snail is essential for disassembly of adherens junctions during epithelial–mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, we track the behavior of E-cadherin–rich junction clusters, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. Our findings provide in vivo evidence for mechanosensitivity of cell–cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT. PMID:26754645

  3. Designing and verifying a disassembly line approach to cope with the upsurge of end-of-life vehicles in China.

    PubMed

    Zhang, Chunliang; Chen, Ming

    2018-06-01

    An upsurge of end-of-life vehicles (ELVs) is emerging in China, which means a potential monumental environmental crisis. The approach of disassembly line is expected to be an effective solution to such increasing volumes. Due to the complexity of vehicle product and uncertainties of disassembly processes, a complete set of disassembly line system should be taken into detailed consideration. We have designed and constructed a novel disassembly line using a flexible transition technique with the objective of complete disassembly. Prior to productivity testing, comparative Arena-based simulations on four scenarios have been performed and finally a best scenario is selected. The results show that the guarantee of cycle time is the key to meet the productivity target of 30,000 vehicles for one year. To achieve it, some constructive measures such as forcible entry tools are given. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. The Radiation Assessment Detector (RAD) Investigation

    NASA Astrophysics Data System (ADS)

    Hassler, D. M.; Zeitlin, C.; Wimmer-Schweingruber, R. F.; Böttcher, S.; Martin, C.; Andrews, J.; Böhm, E.; Brinza, D. E.; Bullock, M. A.; Burmeister, S.; Ehresmann, B.; Epperly, M.; Grinspoon, D.; Köhler, J.; Kortmann, O.; Neal, K.; Peterson, J.; Posner, A.; Rafkin, S.; Seimetz, L.; Smith, K. D.; Tyler, Y.; Weigle, G.; Reitz, G.; Cucinotta, F. A.

    2012-09-01

    The Radiation Assessment Detector (RAD) on the Mars Science Laboratory (MSL) is an energetic particle detector designed to measure a broad spectrum of energetic particle radiation. It will make the first-ever direct radiation measurements on the surface of Mars, detecting galactic cosmic rays, solar energetic particles, secondary neutrons, and other secondary particles created both in the atmosphere and in the Martian regolith. The radiation environment on Mars, both past and present, may have implications for habitability and the ability to sustain life. Radiation exposure is also a major concern for future human missions. The RAD instrument combines charged- and neutral-particle detection capability over a wide dynamic range in a compact, low-mass, low-power instrument. These capabilities are required in order to measure all the important components of the radiation environment. RAD consists of the RAD Sensor Head (RSH) and the RAD Electronics Box (REB) integrated together in a small, compact volume. The RSH contains a solid-state detector telescope with three silicon PIN diodes for charged particle detection, a thallium doped Cesium Iodide scintillator, plastic scintillators for neutron detection and anti-coincidence shielding, and the front-end electronics. The REB contains three circuit boards, one with a novel mixed-signal ASIC for processing analog signals and an associated control FPGA, another with a second FPGA to communicate with the rover and perform onboard analysis of science data, and a third board with power supplies and power cycling or "sleep"-control electronics. The latter enables autonomous operation, independent of commands from the rover. RAD is a highly capable and highly configurable instrument that paves the way for future compact energetic particle detectors in space.

  5. Highly Selective Nuclide Removal from the R-Reactor Disassembly Basin at SRS

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

    Pickett, J.B.

    This paper describes the results of a deployment of highly selective ion-exchange resin technologies for the in-situ removal of Cs-137 and Sr-90 from the Savannah River Site (SRS) R-Reactor Disassembly Basin. The deployment was supported by the DOE Office of Science and Technology's (OST, EM-50) National Engineering Technology Laboratory (NETL), as a part of an Accelerated Site Technology Deployment (ASTD) project. The Facilities Decontamination and Decommissioning (FDD) Program at the SRS conducted this deployment as a part of an overall program to deactivate three of the site's five reactor disassembly basins

  6. The Bacterial Actin MamK

    PubMed Central

    Ozyamak, Ertan; Kollman, Justin; Agard, David A.; Komeili, Arash

    2013-01-01

    It is now recognized that actin-like proteins are widespread in bacteria and, in contrast to eukaryotic actins, are highly diverse in sequence and function. The bacterial actin, MamK, represents a clade, primarily found in magnetotactic bacteria, that is involved in the proper organization of subcellular organelles, termed magnetosomes. We have previously shown that MamK from Magnetospirillum magneticum AMB-1 (AMB-1) forms dynamic filaments in vivo. To gain further insights into the molecular mechanisms that underlie MamK dynamics and function, we have now studied the in vitro properties of MamK. We demonstrate that MamK is an ATPase that, in the presence of ATP, assembles rapidly into filaments that disassemble once ATP is depleted. The mutation of a conserved active site residue (E143A) abolishes ATPase activity of MamK but not its ability to form filaments. Filament disassembly depends on both ATPase activity and potassium levels, the latter of which results in the organization of MamK filaments into bundles. These data are consistent with observations indicating that accessory factors are required to promote filament disassembly and for spatial organization of filaments in vivo. We also used cryo-electron microscopy to obtain a high resolution structure of MamK filaments. MamK adopts a two-stranded helical filament architecture, but unlike eukaryotic actin and other actin-like filaments, subunits in MamK strands are unstaggered giving rise to a unique filament architecture. Beyond extending our knowledge of the properties and function of MamK in magnetotactic bacteria, this study emphasizes the functional and structural diversity of bacterial actins in general. PMID:23204522

  7. The Amphipathic Helix of Influenza A Virus M2 Protein Is Required for Filamentous Bud Formation and Scission of Filamentous and Spherical Particles

    PubMed Central

    Roberts, Kari L.; Leser, George P.; Ma, Chunlong

    2013-01-01

    Influenza virus assembles and buds at the infected-cell plasma membrane. This involves extrusion of the plasma membrane followed by scission of the bud, resulting in severing the nascent virion from its former host. The influenza virus M2 ion channel protein contains in its cytoplasmic tail a membrane-proximal amphipathic helix that facilitates the scission process and is also required for filamentous particle formation. Mutation of five conserved hydrophobic residues to alanines within the amphipathic helix (M2 five-point mutant, or 5PM) reduced scission and also filament formation, whereas single mutations had no apparent phenotype. Here, we show that any two of these five residues mutated together to alanines result in virus debilitated for growth and filament formation in a manner similar to 5PM. Growth kinetics of the M2 mutants are approximately 2 logs lower than the wild-type level, and plaque diameter was significantly reduced. When the 5PM and a representative double mutant (I51A-Y52A) were introduced into A/WSN/33 M2, a strain that produces spherical particles, similar debilitation in viral growth occurred. Electron microscopy showed that with the 5PM and the I51A-Y52A A/Udorn/72 and WSN viruses, scission failed, and emerging virus particles exhibited a “beads-on-a-string” morphology. The major spike glycoprotein hemagglutinin is localized within lipid rafts in virus-infected cells, whereas M2 is associated at the periphery of rafts. Mutant M2s were more widely dispersed, and their abundance at the raft periphery was reduced, suggesting that the M2 amphipathic helix is required for proper localization in the host membrane and that this has implications for budding and scission. PMID:23843641

  8. Assembly via disassembly: A case in machine perceptual development

    NASA Technical Reports Server (NTRS)

    Bajcsy, Ruzena K.; Tsikos, Constantine J.

    1989-01-01

    First results in the effort of learning about representations of objects is presented. The questions attempted to be answered are: What is innate and what must be derived from the environment. The problem is casted in the framework of disassembly of an object into two parts.

  9. AGR-2 Irradiated Test Train Preliminary Inspection and Disassembly First Look

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

    Ploger, Scott; Demkowciz, Paul; Harp, Jason

    2015-05-01

    The AGR 2 irradiation experiment began in June 2010 and was completed in October 2013. The test train was shipped to the Materials and Fuels Complex in July 2014 for post-irradiation examination (PIE). The first PIE activities included nondestructive examination of the test train, followed by disassembly of the test train and individual capsules and detailed inspection of the capsule contents, including the fuel compacts and their graphite fuel holders. Dimensional metrology was then performed on the compacts, graphite holders, and steel capsule shells. AGR 2 disassembly and metrology were performed with the same equipment used successfully on AGR 1more » test train components. Gamma spectrometry of the intact test train gave a preliminary look at the condition of the interior components. No evidence of damage to compacts or graphite components was evident from the isotopic and gross gamma scans. Disassembly of the AGR 2 test train and its capsules was conducted rapidly and efficiently by employing techniques refined during the AGR 1 disassembly campaign. Only one major difficulty was encountered while separating the test train into capsules when thermocouples (of larger diameter than used in AGR 1) and gas lines jammed inside the through tubes of the upper capsules, which required new tooling for extraction. Disassembly of individual capsules was straightforward with only a few minor complications. On the whole, AGR 2 capsule structural components appeared less embrittled than their AGR 1 counterparts. Compacts from AGR 2 Capsules 2, 3, 5, and 6 were in very good condition upon removal. Only relatively minor damage or markings were visible using high resolution photographic inspection. Compact dimensional measurements indicated radial shrinkage between 0.8 to 1.7%, with the greatest shrinkage observed on Capsule 2 compacts that were irradiated at higher temperature. Length shrinkage ranged from 0.1 to 0.9%, with by far the lowest axial shrinkage on Capsule 3

  10. Disassembly factories for electrical and electronic products to recover resources in product and material cycles.

    PubMed

    Basdere, Bahadir; Seliger, Guenther

    2003-12-01

    Cycle economy as a paradigm for industry in the 21st century depends on the economical and ecological treatment of limited resources. The objective is to achieve more use with fewer resources to increase the use-productivity of these resources. The European Union, aware of the adverse environmental impacts associated with electrical and electronic consumer goods in particular, has passed legislation regulating their appropriate end-of-life treatment. Adaptation processes, including essential disassembly and re-assembly operations, contribute significantly toward the economical fulfillment of these new legal requirements. Typically, the disassembly of used products is characterized by a high rate of manual operations, wide variety of product types, and unknown product properties. To cope with such demands, life cycle units or product accompanying information systems, are being developed and used for acquiring data about a specific product throughout its life cycle to aid in determining the level of product deterioration. Modular disassembly processes and tools have been developed and realized to enable the handling of multiple productvariants. They are being implemented in prototypical hybrid disassembly systemsfor large- and small-size electrical and electronic consumer goods.

  11. A penny-shaped crack in a filament reinforced matrix. 1: The filament model

    NASA Technical Reports Server (NTRS)

    Erdogan, F.; Pacella, A. H.

    1973-01-01

    The electrostatic problem of a penny-shaped crack in an elastic matrix which reinforced by filaments or fibers perpendicular to the plane of the crack was studied. The elastic filament model was developed for application to evaluation studies of the stress intensity factor along the periphery of the crack, the stresses in the filaments or fibers, and the interface shear between the matrix and the filaments or fibers. The requirements expected of the model are a sufficiently accurate representation of the filament and applicability to the interaction problems involving a cracked elastic continuum with multi-filament reinforcements. The technique for developing the model and numerical examples of it are shown.

  12. RadNet Air Data From Honolulu, HI

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  13. RadNet Air Data From Birmingham, AL

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  14. RadNet Air Data From Dallas, TX

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  15. RadNet Air Data From Omaha, NE

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  16. RadNet Air Data From Montgomery, AL

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  17. RadNet Air Data From Burlington, VT

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  18. RadNet Air Data From Washington, DC

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  19. RadNet Air Data From Rochester, NY

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  20. RadNet Air Data From Tampa, FL

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  1. RadNet Air Data From Cincinnati, OH

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  2. RadNet Air Data From Fairbanks, AK

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  3. RadNet Air Data From Yuma, AZ

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  5. RadNet Air Data From Kearney, NE

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  6. RadNet Air Data From Phoenix, AZ

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  7. RadNet Air Data From Pierre, SD

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  8. RadNet Air Data From Augusta, GA

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  9. RadNet Air Data From Syracuse, NY

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  10. RadNet Air Data From Albany, NY

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  11. RadNet Air Data From Anchorage, AK

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  12. RadNet Air Data From Philadelphia, PA

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  13. RadNet Air Data From Houston, TX

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  14. RadNet Air Data From Duluth, MN

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  15. RadNet Air Data From Raleigh, NC

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  16. RadNet Air Data From Louisville, KY

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  17. RadNet Air Data From Cleveland, OH

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  18. RadNet Air Data From Carlsbad, NM

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  19. RadNet Air Data From Corvallis, OR

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  20. RadNet Air Data From Orono, ME

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  1. RadNet Air Data From Reno, NV

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  2. RadNet Air Data From Nashville, TN

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  3. RadNet Air Data From Concord, NH

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  4. RadNet Air Data From Paducah, KY

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  5. RadNet Air Data From Edison, NJ

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  6. RadNet Air Data From Wilmington, NC

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  7. RadNet Air Data From Boise, ID

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  8. RadNet Air Data From Albuquerque, NM

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  9. RadNet Air Data From Fresno, CA

    EPA Pesticide Factsheets

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  10. RadNet Air Data From Amarillo, TX

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  11. RadNet Air Data From Portland, OR

    EPA Pesticide Factsheets

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  12. RadNet Air Data From Jacksonville, FL

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  13. RadNet Air Data From Dover, DE

    EPA Pesticide Factsheets

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  14. RadNet Air Data From Baltimore, MD

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  15. RadNet Air Data From Miami, FL

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  16. RadNet Air Data From Billings, MT

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  17. RadNet Air Data From Providence, RI

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  18. RadNet Air Data From Knoxville, TN

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  19. RadNet Air Data From Columbus, OH

    EPA Pesticide Factsheets

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  20. RadNet Air Data From Bloomsburg, PA

    EPA Pesticide Factsheets

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  1. RadNet Air Data From Shreveport, LA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Shreveport, LA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  2. RadNet Air Data From Laredo, TX

    EPA Pesticide Factsheets

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  3. RadNet Air Data From Bakersfield, CA

    EPA Pesticide Factsheets

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  4. RadNet Air Data From Portland, ME

    EPA Pesticide Factsheets

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  5. RadNet Air Data From Champaign, IL

    EPA Pesticide Factsheets

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  6. RadNet Air Data From Tucson, AZ

    EPA Pesticide Factsheets

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  7. RadNet Air Data From Juneau, AK

    EPA Pesticide Factsheets

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  8. RadNet Air Data From Toledo, OH

    EPA Pesticide Factsheets

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  9. RadNet Air Data From Boston, MA

    EPA Pesticide Factsheets

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  10. RadNet Air Data From Indianapolis, IN

    EPA Pesticide Factsheets

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  11. RadNet Air Data From Yaphank, NY

    EPA Pesticide Factsheets

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  12. RadNet Air Data From Anaheim, CA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Anaheim, CA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  13. RadNet Air Data From Riverside, CA

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    This page presents radiation air monitoring and air filter analysis data for Riverside, CA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  14. RadNet Air Data From Detroit, MI

    EPA Pesticide Factsheets

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  15. RadNet Air Data From Wichita, KS

    EPA Pesticide Factsheets

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  16. RadNet Air Data From Columbia, SC

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Columbia, SC from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  17. RadNet Air Data From Milwaukee, WI

    EPA Pesticide Factsheets

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  18. RadNet Air Data From Richmond, VA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Richmond, VA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  19. RadNet Air Data From Tulsa, OK

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Tulsa, OK from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  20. RadNet Air Data From Aurora, IL

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Aurora, IL from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  1. RadNet Air Data From Hartford, CT

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Hartford. CT from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  2. RadNet Air Data From Charleston, WV

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Charleston, WV from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  3. RadNet Air Data From Shawano, WI

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Shawano, WI from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  4. RadNet Air Data From Harlingen, TX

    EPA Pesticide Factsheets

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  5. RadNet Air Data From Springfield, MO

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Springfield, MO from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  6. RadNet Air Data From Olympia, WA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Olympia, WA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  7. RadNet Air Data From Memphis, TN

    EPA Pesticide Factsheets

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  8. RadNet Air Data From Lubbock, TX

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Lubbock, TX from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  9. RadNet Air Data From Sacramento, CA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Sacramento, CA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  10. RadNet Air Data From Lockport, NY

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Lockport, NY from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  11. RadNet Air Data From Jackson, MS

    EPA Pesticide Factsheets

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  12. RadNet Air Data From Seattle, WA

    EPA Pesticide Factsheets

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  13. RadNet Air Data From Pittsburgh, PA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Pittsburgh, PA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  14. RadNet Air Data From Madison, WI

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Madison, WI from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  15. RadNet Air Data From Ellensburg, WA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Ellensburg, WA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  16. RadNet Air Data From Harrisonburg, VA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Harrisonburg, VA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  17. RadNet Air Data From Bismarck, ND

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Bismarck, ND from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  18. RadNet Air Data From Denver, CO

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Denver, CO from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  19. RadNet Air Data From Charlotte, NC

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Charlotte, NC from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  20. RadNet Air Data From Lexington, KY

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    This page presents radiation air monitoring and air filter analysis data for Lexington, KY from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  1. RadNet Air Data From Casper, WY

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Casper, WY from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  2. RadNet Air Data From Eureka, CA

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  3. RadNet Air Data From Lincoln, NE

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Lincoln, NE from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  4. RadNet Air Data From Orlando, FL

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  5. RadNet Air Data From Mobile, AL

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    This page presents radiation air monitoring and air filter analysis data for Mobile, AL from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  6. RadNet Air Data From Spokane, WA

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    This page presents radiation air monitoring and air filter analysis data for Spokane, WA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  7. RadNet Air Data From Atlanta, GA

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    This page presents radiation air monitoring and air filter analysis data for Atlanta, GA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  8. RadNet Air Data From Greensboro, NC

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    This page presents radiation air monitoring and air filter analysis data for Greensboro, NC from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  9. RadNet Air Data From Chicago, IL

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Chicago, IL from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  10. RadNet Air Data From Worcester, MA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Worcester, MA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  11. RadNet Air Data From Austin, TX

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Austin, TX from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  12. Disassembly and physical separation of electric/electronic components layered in printed circuit boards (PCB).

    PubMed

    Lee, Jaeryeong; Kim, Youngjin; Lee, Jae-chun

    2012-11-30

    Although printed circuit boards (PCBs) contain various elements, only the major elements (i.e., those with content levels in wt% or over grade) of and precious metals (e.g., Ag, Au, and platinum groups) contained within PCBs can be recycled. To recover other elements from PCBs, the PCBs should be properly disassembled as the first step of the recycling process. The recovery of these other elements would be beneficial for efforts to conserve scarce resources, reuse electric/electronic components (EECs), and eliminate environmental problems. This paper examines the disassembly of EECs from wasted PCBs (WPCBs) and the physical separation of these EECs using a self-designed disassembling apparatus and a 3-step separation process of sieving, magnetic separation, and dense medium separation. The disassembling efficiencies were evaluated by using the ratio of grinding area (E(area)) and the weight ratio of the detached EECs (E(weight)). In the disassembly treatment, these efficiencies were improved with an increase of grinder speed and grinder height. 97.7% (E(area)) and 98% (E(weight)) could be accomplished ultimately by 3 repetitive treatments at a grinder speed of 5500 rpm and a grinder height of 1.5mm. Through a series of physical separations, most groups of the EECs (except for the diode, transistor, and IC chip groups) could be sorted at a relatively high separation efficiency of about 75% or more. To evaluate the separation efficiency with regard to the elemental composition, the distribution ratio (R(dis)) and the concentration ratio (R(conc)) were used. 15 elements could be separated with the highest R(dis) and R(conc) in the same separated division. This result implies that the recyclability of the elements is highly feasible, even though the initial content in EECs is lower than several tens of mg/kg. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Filamentary structures in dense plasma focus: Current filaments or vortex filaments?

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

    Soto, Leopoldo, E-mail: lsoto@cchen.cl; Pavez, Cristian; Moreno, José

    2014-07-15

    Recent observations of an azimuthally distributed array of sub-millimeter size sources of fusion protons and correlation between extreme ultraviolet (XUV) images of filaments with neutron yield in PF-1000 plasma focus have re-kindled interest in their significance. These filaments have been described variously in literature as current filaments and vortex filaments, with very little experimental evidence in support of either nomenclature. This paper provides, for the first time, experimental observations of filaments on a table-top plasma focus device using three techniques: framing photography of visible self-luminosity from the plasma, schlieren photography, and interferometry. Quantitative evaluation of density profile of filaments frommore » interferometry reveals that their radius closely agrees with the collision-less ion skin depth. This is a signature of relaxed state of a Hall fluid, which has significant mass flow with equipartition between kinetic and magnetic energy, supporting the “vortex filament” description. This interpretation is consistent with empirical evidence of an efficient energy concentration mechanism inferred from nuclear reaction yields.« less

  14. The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress.

    PubMed

    Milbradt, Jens; Hutterer, Corina; Bahsi, Hanife; Wagner, Sabrina; Sonntag, Eric; Horn, Anselm H C; Kaufer, Benedikt B; Mori, Yasuko; Sticht, Heinrich; Fossen, Torgils; Marschall, Manfred

    2016-08-01

    The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear

  15. The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress

    PubMed Central

    Milbradt, Jens; Hutterer, Corina; Bahsi, Hanife; Wagner, Sabrina; Sonntag, Eric; Kaufer, Benedikt B.; Mori, Yasuko; Sticht, Heinrich; Fossen, Torgils; Marschall, Manfred

    2016-01-01

    The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear

  16. Rad-deletion Phenocopies Tonic Sympathetic Stimulation of the Heart

    PubMed Central

    Levitan, Bryana M.; Manning, Janet R.; Withers, Catherine N.; Smith, Jeffrey D.; Shaw, Robin M.; Andres, Douglas A.; Sorrell, Vincent L.

    2016-01-01

    Sympathetic stimulation modulates L-type calcium channel (LTCC) gating to contribute to increased systolic heart function. Rad is a monomeric G-protein that interacts with LTCC. Genetic deletion of Rad (Rad−/−) renders LTCC in a sympathomimetic state. The study goal was to use a clinically inspired pharmacological stress echocardiography test, including analysis of global strain, to determine whether Rad−/− confers tonic positive inotropic heart function. Sarcomere dynamics and strain showed partial parallel isoproterenol (ISO) responsiveness for wild-type (WT) and for Rad−/−. Rad−/− basal inotropy was elevated compared to WT but was less responsiveness to ISO. Rad protein levels were lower in human patients with end-stage non-ischemic heart failure. These results show that Rad reduction provides a stable inotropic response rooted in sarcomere level function. Thus, reduced Rad levels in heart failure patients may be a compensatory response to need for increased output in the setting of HF. Rad deletion suggests a future therapeutic direction for inotropic support. PMID:27798760

  17. Sliding movement of single actin filaments on one-headed myosin filaments

    NASA Astrophysics Data System (ADS)

    Harada, Yoshie; Noguchi, Akira; Kishino, Akiyoshi; Yanagida, Toshio

    1987-04-01

    The myosin molecule consists of two heads, each of which contains an enzymatic active site and an actin-binding site. The fundamental problem of whether the two heads function independently or cooperatively during muscle contraction has been studied by methods using an actomyosin thread1, superprecipitation2-4 and chemical modification of muscle fibres5. No clear conclusion has yet been reached. We have approached this question using an assay system in which sliding movements of fluorescently labelled single actin filaments along myosin filaments can be observed directly6,7. Here, we report direct measurement of the sliding of single actin filaments along one-headed myosin filaments in which the density of heads was varied over a wide range. Our results show that cooperative interaction between the two heads of myosin is not essential for inducing the sliding movement of actin filaments.

  18. Alignment Pins for Assembling and Disassembling Structures

    NASA Technical Reports Server (NTRS)

    Campbell, Oliver C.

    2008-01-01

    Simple, easy-to-use, highly effective tooling has been devised for maintaining alignment of bolt holes in mating structures during assembly and disassembly of the structures. The tooling was originally used during removal of a body flap from the space shuttle Atlantis, in which misalignments during removal of the last few bolts could cause the bolts to bind in their holes. By suitably modifying the dimensions of the tooling components, the basic design of the tooling can readily be adapted to other structures that must be maintained in alignment. The tooling includes tapered, internally threaded alignment pins designed to fit in the bolt holes in one of the mating structures, plus a draw bolt and a cup that are used to install or remove each alignment pin. In preparation for disassembly of two mating structures, external supports are provided to prevent unintended movement of the structures. During disassembly of the structures, as each bolt that joins the structures is removed, an alignment pin is installed in its place. Once all the bolts have been removed and replaced with pins, the pins maintain alignment as the structures are gently pushed or pulled apart on the supports. In assembling the two structures, one reverses the procedure described above: pins are installed in the bolt holes, the structures are pulled or pushed together on the supports, then the pins are removed and replaced with bolts. The figure depicts the tooling and its use. To install an alignment pin in a bolt hole in a structural panel, the tapered end of the pin is inserted from one side of the panel, the cup is placed over the pin on the opposite side of the panel, the draw bolt is inserted through the cup and threaded into the pin, the draw bolt is tightened to pull the pin until the pin is seated firmly in the hole, then the draw bolt and cup are removed, leaving the pin in place. To remove an alignment pin, the cup is placed over the pin on the first-mentioned side of the panel, the draw

  19. Transition of Femtosecond-Filament-Solid Interactions from Single to Multiple Filament Regime

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

    Skrodzki, P. J.; Burger, M.; Jovanovic, I.

    High-peak-power fs-laser filaments offer unique characteristics attractive to remote sensing via techniques such as remote laser-induced breakdown spectroscopy (R-LIBS). The dynamics of several ablation mechanisms following the interaction between a filament and a solid determines the emission strength and reproducibility of target plasma, which is of relevance for R-LIBS applications. Here, we investigate the space- and time-resolved dynamics of ionic and atomic emission from copper as well as the surrounding atmosphere in order to understand limitations of fs-filament-ablation for standoff energy delivery. Furthermore, we probe the shock front produced from filament-target interaction using time-resolved shadowgraphy and infer laser-material coupling efficienciesmore » for both single and multiple filament regimes through analysis of shock expansion with the Sedov model for point detonation. The results provide insight into plasma structure for the range of peak powers up to 30 times the critical power for filamentation P cr. Despite the stochastic nucleation of multiple filaments at peak-powers greater than 16 P cr, emission of ionic and neutral species increases with pump beam intensity, and short-lived nitrogen emission originating from the ambient is consistently observed. Ultimately, results suggest favorable scaling of emission intensity from target species on the laser pump energy, furthering the prospects for use of filament-solid interactions for remote sensing.« less

  20. Transition of Femtosecond-Filament-Solid Interactions from Single to Multiple Filament Regime

    DOE PAGES

    Skrodzki, P. J.; Burger, M.; Jovanovic, I.

    2017-10-06

    High-peak-power fs-laser filaments offer unique characteristics attractive to remote sensing via techniques such as remote laser-induced breakdown spectroscopy (R-LIBS). The dynamics of several ablation mechanisms following the interaction between a filament and a solid determines the emission strength and reproducibility of target plasma, which is of relevance for R-LIBS applications. Here, we investigate the space- and time-resolved dynamics of ionic and atomic emission from copper as well as the surrounding atmosphere in order to understand limitations of fs-filament-ablation for standoff energy delivery. Furthermore, we probe the shock front produced from filament-target interaction using time-resolved shadowgraphy and infer laser-material coupling efficienciesmore » for both single and multiple filament regimes through analysis of shock expansion with the Sedov model for point detonation. The results provide insight into plasma structure for the range of peak powers up to 30 times the critical power for filamentation P cr. Despite the stochastic nucleation of multiple filaments at peak-powers greater than 16 P cr, emission of ionic and neutral species increases with pump beam intensity, and short-lived nitrogen emission originating from the ambient is consistently observed. Ultimately, results suggest favorable scaling of emission intensity from target species on the laser pump energy, furthering the prospects for use of filament-solid interactions for remote sensing.« less

  1. The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage.

    PubMed

    Vialard, J E; Gilbert, C S; Green, C M; Lowndes, N F

    1998-10-01

    The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells. Treatment with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein. All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage. Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.

  2. Safety and Immunogenicity of a rAd35-EnvA Prototype HIV-1 Vaccine in Combination with rAd5-EnvA in Healthy Adults (VRC 012).

    PubMed

    Crank, Michelle C; Wilson, Eleanor M P; Novik, Laura; Enama, Mary E; Hendel, Cynthia S; Gu, Wenjuan; Nason, Martha C; Bailer, Robert T; Nabel, Gary J; McDermott, Adrian B; Mascola, John R; Koup, Richard A; Ledgerwood, Julie E; Graham, Barney S

    2016-01-01

    VRC 012 was a Phase I study of a prototype recombinant adenoviral-vector serotype-35 (rAd35) HIV vaccine, the precursor to two recently published clinical trials, HVTN 077 and 083. On the basis of prior evaluation of multiclade rAd5 HIV vaccines, Envelope A (EnvA) was selected as the standard antigen for a series of prototype HIV vaccines to compare various vaccine platforms. In addition, prior studies of rAd5-vectored vaccines suggested pre-existing human immunity may be a confounding factor in vaccine efficacy. rAd35 is less seroprevalent across human populations and was chosen for testing alone and in combination with a rAd5-EnvA vaccine in the present two-part phase I study. First, five subjects each received a single injection of 109, 1010, or 1011 particle units (PU) of rAd35-EnvA in an open-label, dose-escalation study. Next, 20 Ad5/Ad35-seronegative subjects were randomized to blinded, heterologous prime-boost schedules combining rAd5-EnvA and rAd35-EnvA with a three month interval. rAd35-EnvA was given at 1010 or 1011 PU to ten subjects each; all rAd5-EnvA injections were 1010 PU. EnvA-specific immunogenicity was assessed four weeks post-injection. Solicited reactogenicity and clinical safety were followed after each injection. Vaccinations were well tolerated at all dosages. Antibody responses measured by ELISA were detected at 4 weeks in 30% and 50% of subjects after single doses of 1010 or 1011 PU rAd35, respectively, and in 89% after a single rAd5-EnvA 1010 PU injection. EnvA-specific IFN-γ ELISpot responses were detected at four weeks in 0%, 70%, and 50% of subjects after the respective rAd35-EnvA dosages compared to 89% of subjects after rAd5. T cell responses were higher after a single rAd5-EnvA 1010 PU injection than after a single rAd35-EnvA 1010 PU injection, and humoral responses were low after a single dose of either vector. Of those completing the vaccine schedule, 100% of rAd5-EnvA recipients and 90% of rAd35-EnvA recipients had both T cell

  3. Galactic cold cores. VIII. Filament formation and evolution: Filament properties in context with evolutionary models

    NASA Astrophysics Data System (ADS)

    Rivera-Ingraham, A.; Ristorcelli, I.; Juvela, M.; Montillaud, J.; Men'shchikov, A.; Malinen, J.; Pelkonen, V.-M.; Marston, A.; Martin, P. G.; Pagani, L.; Paladini, R.; Paradis, D.; Ysard, N.; Ward-Thompson, D.; Bernard, J.-P.; Marshall, D. J.; Montier, L.; Tóth, L. V.

    2017-05-01

    Context. The onset of star formation is intimately linked with the presence of massive unstable filamentary structures. These filaments are therefore key for theoretical models that aim to reproduce the observed characteristics of the star formation process in the Galaxy. Aims: As part of the filament study carried out by the Herschel Galactic Cold Cores Key Programme, here we study and discuss the filament properties presented in GCC VII (Paper I) in context with theoretical models of filament formation and evolution. Methods: A conservatively selected sample of filaments located at a distance D< 500 pc was extracted from the GCC fields with the getfilaments algorithm. The physical structure of the filaments was quantified according to two main components: the central (Gaussian) region of the filament (core component), and the power-law-like region dominating the filament column density profile at larger radii (wing component). The properties and behaviour of these components relative to the total linear mass density of the filament and the column density of its environment were compared with the predictions from theoretical models describing the evolution of filaments under gravity-dominated conditions. Results: The feasibility of a transition from a subcritical to supercritical state by accretion at any given time is dependent on the combined effect of filament intrinsic properties and environmental conditions. Reasonably self-gravitating (high Mline,core) filaments in dense environments (AV≳ 3 mag) can become supercritical on timescales of t 1 Myr by accreting mass at constant or decreasing width. The trend of increasing Mline,tot (Mline,core and Mline,wing) and ridge AV with background for the filament population also indicates that the precursors of star-forming filaments evolve coevally with their environment. The simultaneous increase of environment and filament AV explains the observed association between dense environments and high Mline,core values

  4. Highly Selective Nuclide Removal from the R-Reactor Disassembly Basin at the SRS

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

    Pickett, J. B.; Austin, W. E.; Dukes, H. H.

    This paper describes the results of a deployment of highly selective ion-exchange resin technologies for the in-situ removal of Cs-137 and Sr-90 from the Savannah River Site (SRS) R-Reactor Disassembly Basin. The deployment was supported by the DOE Office of Science and Technology's (OST, EM-50) National Engineering Technology Laboratory (NETL), as a part of an Accelerated Site Technology Deployment (ASTD) project. The Facilities Decontamination and Decommissioning (FDD) Program at the SRS conducted this deployment as a part of an overall program to deactivate three of the site's five reactor disassembly basins.

  5. A Summary Report on the NPH Evaluation of 105-L Disassembly Basin

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

    Joshi, J.R.

    2002-04-30

    The L Area Disassembly Basin (LDB) is evaluated for the natural phenomena hazards (NPH) effects due to earthquake, wind, and tornado in accordance with DOE Order 420.1 and DOE-STD-1020. The deterministic analysis is performed for a Performance Category 3 (PC3) level of loads. Savannah River Site (SRS) specific NPH loads and design criteria are obtained from Engineering Standard 01060. It is demonstrated that the demand to capacity (D/C) ratios for primary and significant structural elements are acceptable (equal to or less than 1.0). Thus, 105-L Disassembly Basin building structure is qualified for the PC3 NPH effects in accordance with DOEmore » Order 420.1.« less

  6. Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers

    PubMed Central

    Kiuchi, Tai; Ohashi, Kazumasa; Kurita, Souichi; Mizuno, Kensaku

    2007-01-01

    Cofilin stimulates actin filament disassembly and accelerates actin filament turnover. Cofilin is also involved in stimulus-induced actin filament assembly during lamellipodium formation. However, it is not clear whether this occurs by replenishing the actin monomer pool, through filament disassembly, or by creating free barbed ends, through its severing activity. Using photoactivatable Dronpa-actin, we show that cofilin is involved in producing more than half of all cytoplasmic actin monomers and that the rate of actin monomer incorporation into the tip of the lamellipodium is dependent on the size of this actin monomer pool. Finally, in cofilin-depleted cells, stimulus-induced actin monomer incorporation at the cell periphery is attenuated, but the incorporation of microinjected actin monomers is not. We propose that cofilin contributes to stimulus-induced actin filament assembly and lamellipodium extension by supplying an abundant pool of cytoplasmic actin monomers. PMID:17470633

  7. [The preparation of recombinant adenovirus Ad-Rad50-GFP and detection of the optimal multiplicity of infection in CNE1 transfected hv Ad-Rad50-GFP].

    PubMed

    Yan, Ruicheng; Huang, Jiancong; Zhu, Ling; Chang, Lihong; Li, Jingjia; Wu, Xifu; Ye, Jin; Zhang, Gehua

    2015-12-01

    The optimal multiplicity of infection (MOI) of the recombinant adenovirus Ad-Rad50-GFP carrying a mutant Rad50 gene expression region on the cell growth of nasopharyngeal carcinoma and the viral amplification efficiency of CNE1 cell infected by this adenovirus were studied. The biological titer of Ad-Rad50-GFP was measured by end point dilution method. The impact of recombinant adenoviral vector transfection on the growth of CNE1 cells was observed by cell growth curve. Transfection efficacy of recombinant adenoviral vector was observed and calculated through fluorescence microscope. The expression f mutant Rad50 in the Ad-Rad50-GFP transfected CNE1 cells with optimal MOI was detected by Western Blot after transfection. The biological titer of Ad-Rad50-GFP was 1.26 x 10¹¹ pfu/ml. CNE1 cell growth was not influenced significantly as they were transfected by recombinant adenoviral vector with MOI less than 50. Transfection efficacy of recombinant adenoviral vector was most salient at 24 hours after transfection, with the high expression of mutant Rad50, and the efficiency still remained about 70% after 72 hours. Recombinant adenoviral vector Ad-Rad50-GFP could transfect CNE1 cells as well as result in the expression of mutant Rad50 in CNE1 cells effectively. MOI = 50 was the optimal multiplicity of infection of CNE1 cells transfected by recombinant adenoviral vector Ad-Rad50-GFP.

  8. Dual-responsive polyplexes with enhanced disassembly and endosomal escape for efficient delivery of siRNA.

    PubMed

    Zhu, Jia; Qiao, Mingxi; Wang, Qi; Ye, Yuqing; Ba, Shuang; Ma, Jingjing; Hu, Haiyang; Zhao, Xiuli; Chen, Dawei

    2018-04-01

    Despite the extracellular barriers for siRNA delivery have been overcome by utilizing advanced nanoparticle delivery systems, the key intracellular barriers after internalization including efficient disassembly of siRNA and endosomal escape still remains challenging. To address the issues, we developed a unique pH- and redox potential-responsive polyplex delivery system based on the copolymer of mPEG-b-PLA-PHis-ssPEI1.8 k, which is composed of a pH-responsive copolymer of PEG-b-PLA-PHis (Mw 5 k) and a branched PEI (Mw1.8 k) linked with redox cleavable disulfide bond. The copolymer showed excellent siRNA complexation and protection abilities against endogenous substances at the relatively low N/P ratio of 6. The siRNA release from the polyplexes (N/P 6) was markedly increased from 13.62% to 58.67% under conditions simulating the endosomal microenvironment. Fluorescence resonance energy transfer (FRET) test also indicated a higher disassembly extent of siRNA from the copolymer. The accelerated siRNA release from the polyplexes was markedly restrained when the N/P ratio was raised above 10 due to the increasing of electrostatic interactions. The efficient endosomal escape of siRNA after internalization was confirmed by confocal microscopy, which was attributed to the cleavaged PEI chains inducing membrane destabilization, the "proton sponge effect" of PHis and PEI as well as the relative small size of after disassembly. The enhanced disassembly and endosomal escape were elucidated as the leading cause for polyplexes (N/P 6) showed more efficient Bcl-2 silencing (85.45%) than those polyplexes with higher N/P ratios (N/P 10 and 15). In vivo results further demonstrated that polyplexes (N/P 6) delivery of siBcl-2 significantly inhibited the MCF-7 breast tumor growth as compared to its counterparts. The incorporation of convertible non-electrical interactions at a balance with electrostatic interactions in complexation siRNA has been demonstrated as an effective

  9. Protective role of RAD50 on chromatin bridges during abnormal cytokinesis.

    PubMed

    Schröder-Heurich, Bianca; Wieland, Britta; Lavin, Martin F; Schindler, Detlev; Dörk, Thilo

    2014-03-01

    Faithful chromosome segregation is required for preserving genomic integrity. Failure of this process may entail chromatin bridges preventing normal cytokinesis. To test whether RAD50, a protein normally involved in DNA double-strand break repair, is involved in abnormal cytokinesis and formation of chromatin bridges, we used immunocytochemical and protein interaction assays. RAD50 localizes to chromatin bridges during aberrant cytokinesis and subsequent stages of the cell cycle, either decorating the entire bridge or focally accumulating at the midbody zone. Ionizing radiation led to an ∼4-fold increase in the rate of chromatin bridges in an ataxia telangiectatica mutated (ATM)-dependent manner in human RAD50-proficient fibroblasts but not in RAD50-deficient cells. Cells with a RAD50-positive chromatin bridge were able to continue cell cycling and to progress through S phase (44%), whereas RAD50 knockdown caused a deficiency in chromatin bridges as well as an ∼4-fold prolonged duration of mitosis. RAD50 colocalized and directly interacted with Aurora B kinase and phospho-histone H3, and Aurora B kinase inhibition led to a deficiency in RAD50-positive bridges. Based on these observations, we propose that RAD50 is a crucial factor for the stabilization and shielding of chromatin bridges. Our study provides evidence for a hitherto unknown role of RAD50 in abnormal cytokinesis.

  10. Mongoose: Creation of a Rad-Hard MIPS R3000

    NASA Technical Reports Server (NTRS)

    Lincoln, Dan; Smith, Brian

    1993-01-01

    This paper describes the development of a 32 Bit, full MIPS R3000 code-compatible Rad-Hard CPU, code named Mongoose. Mongoose progressed from contract award, through the design cycle, to operational silicon in 12 months to meet a space mission for NASA. The goal was the creation of a fully static device capable of operation to the maximum Mil-883 derated speed, worst-case post-rad exposure with full operational integrity. This included consideration of features for functional enhancements relating to mission compatibility and removal of commercial practices not supported by Rad-Hard technology. 'Mongoose' developed from an evolution of LSI Logic's MIPS-I embedded processor, LR33000, code named Cobra, to its Rad-Hard 'equivalent', Mongoose. The term 'equivalent' is used to infer that the core of the processor is functionally identical, allowing the same use and optimizations of the MIPS-I Instruction Set software tool suite for compilation, software program trace, etc. This activity was started in September of 1991 under a contract from NASA-Goddard Space Flight Center (GSFC)-Flight Data Systems. The approach affected a teaming of NASA-GSFC for program development, LSI Logic for system and ASIC design coupled with the Rad-Hard process technology, and Harris (GASD) for Rad-Hard microprocessor design expertise. The program culminated with the generation of Rad-Hard Mongoose prototypes one year later.

  11. Teaching Assembly for Disassembly; An Under-Graduate Module Experience

    ERIC Educational Resources Information Center

    Alexandri, Eleftheria

    2014-01-01

    This paper is about the experience of teaching Assembly for Disassembly to fourth year architect students within the module of sustainable design. When designing a sustainable building one should take into consideration the fact that the building is going to be demolished in some years; thus the materials should be assembled in such a way so that…

  12. Find RAD Partner Programs

    EPA Pesticide Factsheets

    RAD partner programs help protect the ozone layer and reduce emissions of greenhouse gases by disposing of older, inefficient refrigerated appliances using the best environmental practices and technologies available.

  13. Assembly and disassembly of the nucleolus during the cell cycle.

    PubMed

    Hernandez-Verdun, Danièle

    2011-01-01

    The nucleolus is a large nuclear domain in which transcription, maturation and assembly of ribosomes take place. In higher eukaryotes, nucleolar organization in three sub-domains reflects the compartmentation of the machineries related to active or inactive transcription of the ribosomal DNA, ribosomal RNA processing and assembly with ribosomal proteins of the two (40S and 60S) ribosomal subunits. The assembly of the nucleoli during telophase/early G(1) depends on pre-existing machineries inactivated during prophase (the transcription machinery and RNP processing complexes) and on partially processed 45S rRNAs inherited throughout mitosis. In telophase, the 45S rRNAs nucleate the prenucleolar bodies and order the dynamics of nucleolar assembly. The assembly/disassembly processes of the nucleolus depend on the equilibrium between phosphorylation/dephosphorylation of the transcription machinery and on the RNP processing complexes under the control of the CDK1-cyclin B kinase and PP1 phosphatases. The dynamics of assembly/disassembly of the nucleolus is time and space regulated.

  14. pH-Sensitive Reversible Programmed Targeting Strategy by the Self-Assembly/Disassembly of Gold Nanoparticles.

    PubMed

    Ma, Jinlong; Hu, Zhenpeng; Wang, Wei; Wang, Xinyu; Wu, Qiang; Yuan, Zhi

    2017-05-24

    A reversible programmed targeting strategy could achieve high tumor accumulation due to its long blood circulation time and high cellular internalization. Here, targeting ligand-modified poly(ethylene glycol) (PEG-ligand), dibutylamines (Bu), and pyrrolidinamines (Py) were introduced on the surface of gold nanoparticles (Au NPs) for reversible shielding/deshielding of the targeting ligands by pH-responsive self-assembly. Hydrophobic interaction and steric repulsion are the main driving forces for the self-assembly/disassembly of Au NPs. The precise self-assembly (pH ≥ 7.2) and disassembly (pH ≤ 6.8) of Au NPs with different ligands could be achieved by fine-tuning the modifying molar ratio of Bu and Py (R m ), which followed the formula R m = 1/(-0.0013X 2 + 0.0323X + 1), in which X is the logarithm of the partition coefficient of the targeting ligand. The assembled/disassembled behavior of Au NPs at pH 7.2 and 6.8 was confirmed by transmission electron microscopy and dynamic light scattering. Enzyme-linked immunosorbent assays and cellular uptake studies showed that the ligands could be buried inside the assembly and exposed when disassembled. More importantly, this process was reversible, which provides the possibility of prolonging blood circulation by shielding ligands associated with the NPs that were effused from tumor tissue.

  15. RadNet Air Data From San Juan, PR

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for San Juan, PR from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  16. RadNet Air Data From Grand Rapids, MI

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Grand Rapids, MI from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  17. RadNet Air Data From Corpus Christi, TX

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Corpus Christi, TX from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  18. RadNet Air Data From Little Rock, AR

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Little Rock, AR from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  19. RadNet Air Data From Des Moines, IA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Des Moines, IA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  20. RadNet Air Data From Fort Madison, IA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Fort Madison, IA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  1. RadNet Air Data From Fort Wayne, IN

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Fort Wayne, IN from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  2. RadNet Air Data From Navajo Lake, NM

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Navajo Lake, NM from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  3. RadNet Air Data From Las Vegas, NV

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Las Vegas, NV from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  4. RadNet Air Data From St. George, UT

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for St. George, UT from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  5. RadNet Air Data From Jefferson City, MO

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Jefferson City, MO from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  6. RadNet Air Data From Fort Worth, TX

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Fort Worth, TX from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  7. RadNet Air Data From Kansas City, KS

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Kansas City, KS from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  8. RadNet Air Data From San Angelo, TX

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for San Angelo, TX from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  9. RadNet Air Data From San Francisco, CA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for San Francisco, CA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  10. RadNet Air Data From Oklahoma City, OK

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Oklahoma City, OK from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  11. RadNet Air Data From San Bernardino, CA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for San Bernardino, CA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  12. RadNet Air Data From Idaho Falls, ID

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Idaho Falls, ID from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  13. RadNet Air Data From Los Angeles, CA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Los Angeles, CA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  14. RadNet Air Data From El Paso, TX

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for El Paso, TX from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  15. RadNet Air Data From Grand Junction, CO

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Grand Junction, CO from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  16. RadNet Air Data From St. Paul, MN

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for St. Paul, MN from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  17. RadNet Air Data From Virginia Beach, VA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Virginia Beach, VA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  18. RadNet Air Data From La Crosse, WI

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for La Crosse, WI from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  19. RadNet Air Data From San Diego, CA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for San Diego, CA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  20. RadNet Air Data From San Jose, CA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for San Jose, CA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  1. RadNet Air Data From San Antonio, TX

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for San Antonio, TX from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  2. RadNet Air Data From Rapid City, SD

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Rapid City, SD from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  3. RadNet Air Data From Dodge City, KS

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Dodge City, KS from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  4. RadNet Air Data From Colorado Springs, CO

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Colorado Springs, CO from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  5. RadNet Air Data From St. Louis, MO

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for St. Louis, MO from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  6. RadNet Air Data From Fort Smith, AR

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Fort Smith, AR from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  7. RadNet Air Data From Bay City, MI

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Bay City, MI from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  8. RadNet Air Data From Mason City, IA

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Mason City, IA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  9. The Saccharomyces cerevisiae Mre11-Rad50-Xrs2 complex promotes trinucleotide repeat expansions independently of homologous recombination.

    PubMed

    Ye, Yanfang; Kirkham-McCarthy, Lucy; Lahue, Robert S

    2016-07-01

    Trinucleotide repeats (TNRs) are tandem arrays of three nucleotides that can expand in length to cause at least 17 inherited human diseases. Somatic expansions in patients can occur in differentiated tissues where DNA replication is limited and cannot be a primary source of somatic mutation. Instead, mouse models of TNR diseases have shown that both inherited and somatic expansions can be suppressed by the loss of certain DNA repair factors. It is generally believed that these repair factors cause misprocessing of TNRs, leading to expansions. Here we extend this idea to show that the Mre11-Rad50-Xrs2 (MRX) complex of Saccharomyces cerevisiae is a causative factor in expansions of short TNRs. Mutations that eliminate MRX subunits led to significant suppression of expansions whereas mutations that inactivate Rad51 had only a minor effect. Coupled with previous evidence, this suggests that MRX drives expansions of short TNRs through a process distinct from homologous recombination. The nuclease function of Mre11 was dispensable for expansions, suggesting that expansions do not occur by Mre11-dependent nucleolytic processing of the TNR. Epistasis between MRX and post-replication repair (PRR) was tested. PRR protects against expansions, so a rad5 mutant gave a high expansion rate. In contrast, the mre11 rad5 double mutant gave a suppressed expansion rate, indistinguishable from the mre11 single mutant. This suggests that MRX creates a TNR substrate for PRR. Protein acetylation was also tested as a mechanism regulating MRX activity in expansions. Six acetylation sites were identified in Rad50. Mutation of all six lysine residues to arginine gave partial bypass of a sin3 HDAC mutant, suggesting that Rad50 acetylation is functionally important for Sin3-mediated expansions. Overall we conclude that yeast MRX helps drive expansions of short TNRs by a mechanism distinct from its role in homologous recombination and independent of the nuclease function of Mre11. Copyright

  10. Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.

    PubMed

    Buisson, Rémi; Dion-Côté, Anne-Marie; Coulombe, Yan; Launay, Hélène; Cai, Hong; Stasiak, Alicja Z; Stasiak, Andrzej; Xia, Bing; Masson, Jean-Yves

    2010-10-01

    Inherited mutations in human PALB2 are associated with a predisposition to breast and pancreatic cancers. PALB2's tumor-suppressing effect is thought to be based on its ability to facilitate BRCA2's function in homologous recombination. However, the biochemical properties of PALB2 are unknown. Here we show that human PALB2 binds DNA, preferentially D-loop structures, and directly interacts with the RAD51 recombinase to stimulate strand invasion, a vital step of homologous recombination. This stimulation occurs through reinforcing biochemical mechanisms, as PALB2 alleviates inhibition by RPA and stabilizes the RAD51 filament. Moreover, PALB2 can function synergistically with a BRCA2 chimera (termed piccolo, or piBRCA2) to further promote strand invasion. Finally, we show that PALB2-deficient cells are sensitive to PARP inhibitors. Our studies provide the first biochemical insights into PALB2's function with piBRCA2 as a mediator of homologous recombination in DNA double-strand break repair.

  11. CYTOPLASMIC FILAMENTS OF AMOEBA PROTEUS

    PubMed Central

    Pollard, Thomas D.; Ito, Susumu

    1970-01-01

    The role of filaments in consistency changes and movement in a motile cytoplasmic extract of Amoeba proteus was investigated by correlating light and electron microscopic observations with viscosity measurements. The extract is prepared by the method of Thompson and Wolpert (1963). At 0°C, this extract is nonmotile and similar in structure to ameba cytoplasm, consisting of groundplasm, vesicles, mitochondria, and a few 160 A filaments. The extract undergoes striking ATP-stimulated streaming when warmed to 22°C. Two phases of movement are distinguished. During the first phase, the apparent viscosity usually increases and numerous 50–70 A filaments appear in samples of the extract prepared for electron microscopy, suggesting that the increase in viscosity in caused, at least in part, by the formation of these thin filaments. During this initial phase of ATP-stimulated movement, these thin filaments are not detectable by phase-contrast or polarization microscopy, but later, in the second phase of movement, 70 A filaments aggregate to form birefringent microscopic fibrils. A preparation of pure groundplasm with no 160 A filaments or membranous organelles exhibits little or no ATP-stimulated movement, but 50–70 A filaments form and aggregate into birefringent fibrils. This observation and the structural relationship of the 70 A and the 160 A filaments in the motile extract suggest that both types of filaments may be required for movement. These two types of filaments, 50–70 A and 160 A, are also present in the cytoplasm of intact amebas. Fixed cells could not be used to study the distribution of these filaments during natural ameboid movement because of difficulties in preserving the normal structure of the ameba during preparation for electron microscopy. PMID:4915451

  12. Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae

    PubMed Central

    Szyjka, Shawn J.; Aparicio, Jennifer G.; Viggiani, Christopher J.; Knott, Simon; Xu, Weihong; Tavaré, Simon; Aparicio, Oscar M.

    2008-01-01

    Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase, which plays a vital role in survival by stabilizing stalled replication forks. However, evidence that Rad53 directly modulates the activity of replication forks has been lacking, and the nature of fork stabilization has remained unclear. Recently, cells lacking the Psy2–Pph3 phosphatase were shown to be defective in dephosphorylation of Rad53 as well as replication fork restart after DNA damage, suggesting a mechanistic link between Rad53 deactivation and fork restart. To test this possibility we examined the progression of replication forks in methyl-methanesulfonate (MMS)-damaged cells, under different conditions of Rad53 activity. Hyperactivity of Rad53 in pph3Δ cells slows fork progression in MMS, whereas deactivation of Rad53, through expression of dominant-negative Rad53-KD, is sufficient to allow fork restart during recovery. Furthermore, combined deletion of PPH3 and PTC2, a second, unrelated Rad53 phosphatase, results in complete replication fork arrest and lethality in MMS, demonstrating that Rad53 deactivation is a key mechanism controlling fork restart. We propose a model for regulation of replication fork progression through damaged DNA involving a cycle of Rad53 activation and deactivation that coordinates replication restart with DNA repair. PMID:18628397

  13. Cytoplasmic filaments of Amoeba proteus. I. The role of filaments in consistency changes and movement.

    PubMed

    Pollard, T D; Ito, S

    1970-08-01

    The role of filaments in consistency changes and movement in a motile cytoplasmic extract of Amoeba proteus was investigated by correlating light and electron microscopic observations with viscosity measurements. The extract is prepared by the method of Thompson and Wolpert (1963). At 0 degrees C, this extract is nonmotile and similar in structure to ameba cytoplasm, consisting of groundplasm, vesicles, mitochondria, and a few 160 A filaments. The extract undergoes striking ATP-stimulated streaming when warmed to 22 degrees C. Two phases of movement are distinguished. During the first phase, the apparent viscosity usually increases and numerous 50-70 A filaments appear in samples of the extract prepared for electron microscopy, suggesting that the increase in viscosity in caused, at least in part, by the formation of these thin filaments. During this initial phase of ATP-stimulated movement, these thin filaments are not detectable by phase-contrast or polarization microscopy, but later, in the second phase of movement, 70 A filaments aggregate to form birefringent microscopic fibrils. A preparation of pure groundplasm with no 160 A filaments or membranous organelles exhibits little or no ATP-stimulated movement, but 50-70 A filaments form and aggregate into birefringent fibrils. This observation and the structural relationship of the 70 A and the 160 A filaments in the motile extract suggest that both types of filaments may be required for movement. These two types of filaments, 50-70 A and 160 A, are also present in the cytoplasm of intact amebas. Fixed cells could not be used to study the distribution of these filaments during natural ameboid movement because of difficulties in preserving the normal structure of the ameba during preparation for electron microscopy.

  14. Characterization of iminothiosulfine-type ions [HNCS 2] rad +/ rad - and their neutral counterparts by mass spectrometry and computational chemistry

    NASA Astrophysics Data System (ADS)

    Vivekananda, S.; Raghunath, P.; Bhanuprakash, K.; Srinivas, R.; Trikoupis, Moschoula A.; Terlouw, Johan K.

    2000-12-01

    Electron ionization of rhodanine yields iminothiosulfine ions H- N C- S- Srad + , 1brad + , which readily communicate with the higher energy cyclic isomer H- N CS2rad + , 1arad + . CBS-QB3 and G AUSSIAN-2 model chemistries predict that one electron reduction reverses the stability order but that the (singlet) neutrals remain connected via a negligible energy barrier. Neutralization-reionization (NR) experiments demonstrate that singlet 1a and its heterocumulene isomer 1b are stable species in the gas-phase. However, the co-generated triplet species readily dissociate into 3S2rad + + HNC. Confirmatory experimental evidence comes from charge reversal (CR) and NR experiments on the cyclic anion H- N CS2rad - , 1arad - .

  15. Human RAD50 makes a functional DNA-binding complex.

    PubMed

    Kinoshita, Eri; van Rossum-Fikkert, Sari; Sanchez, Humberto; Kertokalio, Aryandi; Wyman, Claire

    2015-06-01

    The MRE11-RAD50-NBS1 (MRN) complex has several distinct functions in DNA repair including important roles in both non-homologous end-joining (NHEJ) and homologous recombination (HR). The biochemical activities of MR(N) have been well characterized implying specific functional roles for the components. The arrangement of proteins in the complex implies interdependence of their biochemical activities making it difficult to separate specific functions. We obtained purified human RAD50 and observed that it binds ATP, undergoes ATP-dependent conformational changes as well as having ATPase activity. Scanning force microscopy analysis clearly showed that RAD50 binds DNA although not as oligomers. RAD50 alone was not functional in tethering DNA molecules. ATP increased formation of RAD50 multimers which were however globular lacking extended coiled coils, in contrast to the MR complex where ATP induced oligomers have obvious coiled coils protruding from a central domain. These results suggest that MRE11 is important in maintaining the structural arrangement of RAD50 in the protein complex and perhaps has a role in reinforcing proper alignment of the coiled coils in the ATP-bound state. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  16. Asbestos exposure from gaskets during disassembly of a medium duty diesel engine.

    PubMed

    Liukonen, Larry R; Weir, Francis W

    2005-03-01

    Diesel engines have historically used asbestos-containing gaskets leading to concerns of fiber release and mechanic exposure. Other published studies regarding asbestos fiber release during gasket removal have reported on short-duration events; were conducted under simulated work conditions; or had other limitations. There are no comprehensive studies relating to diesel engine gaskets under conditions similar to those reported herein, evaluating asbestos fiber release from gaskets during all facets of a complete disassembly and cleaning of a medium duty diesel engine in a busy repair and service shop by a journeyman mechanic. Asbestos content of all gaskets was identified; all disassembly tasks were described and timed; and personal and area air monitoring was conducted for each task. Twenty seven of thirty three gaskets contained chrysotile asbestos in concentrations that ranged from 5 to 70%. All but one air monitoring sample reported results below the limit of reliable detection even though plumes of visible dust were evident during various removal, cleaning, and buffing procedures. The detection limit for airborne asbestos fibers in this investigation was influenced by the presence of other shop dust in the air. Our investigation demonstrates that using shop-standard procedures in an established repair facility, a journeyman mechanic has very little potential for exposure to airborne asbestos fibers during disassembly of an engine, approximately 10% or less than that currently considered to be acceptable by OSHA.

  17. Solid friction between soft filaments.

    PubMed

    Ward, Andrew; Hilitski, Feodor; Schwenger, Walter; Welch, David; Lau, A W C; Vitelli, Vincenzo; Mahadevan, L; Dogic, Zvonimir

    2015-06-01

    Any macroscopic deformation of a filamentous bundle is necessarily accompanied by local sliding and/or stretching of the constituent filaments. Yet the nature of the sliding friction between two aligned filaments interacting through multiple contacts remains largely unexplored. Here, by directly measuring the sliding forces between two bundled F-actin filaments, we show that these frictional forces are unexpectedly large, scale logarithmically with sliding velocity as in solid-like friction, and exhibit complex dependence on the filaments' overlap length. We also show that a reduction of the frictional force by orders of magnitude, associated with a transition from solid-like friction to Stokes's drag, can be induced by coating F-actin with polymeric brushes. Furthermore, we observe similar transitions in filamentous microtubules and bacterial flagella. Our findings demonstrate how altering a filament's elasticity, structure and interactions can be used to engineer interfilament friction and thus tune the properties of fibrous composite materials.

  18. Interobserver and intermodality agreement of standardized algorithms for non-invasive diagnosis of hepatocellular carcinoma in high-risk patients: CEUS-LI-RADS versus MRI-LI-RADS.

    PubMed

    Schellhaas, Barbara; Hammon, Matthias; Strobel, Deike; Pfeifer, Lukas; Kielisch, Christian; Goertz, Ruediger S; Cavallaro, Alexander; Janka, Rolf; Neurath, Markus F; Uder, Michael; Seuss, Hannes

    2018-04-19

    We compared the interobserver agreement for the recently introduced contrast-enhanced ultrasound (CEUS)-based algorithm CEUS-LI-RADS (Liver Imaging Reporting and Data System) versus the well-established magnetic resonance imaging (MRI)-LI-RADS for non-invasive diagnosis of hepatocellular carcinoma (HCC) in high-risk patients. Focal liver lesions in 50 high-risk patients (mean age 66.2 ± 11.8 years; 39 male) were assessed retrospectively with CEUS and MRI. Two independent observers reviewed CEUS and MRI examinations, separately, classifying observations according to CEUS-LI-RADSv.2016 and MRI-LI-RADSv.2014. Interobserver agreement was assessed with Cohen's kappa. Forty-three lesions were HCCs; two were intrahepatic cholangiocarcinomas; five were benign lesions. Arterial phase hyperenhancement was perceived less frequently with CEUS than with MRI (37/50 / 38/50 lesions = 74%/78% [CEUS; observer 1/observer 2] versus 46/50 / 44/50 lesions = 92%/88% [MRI; observer 1/observer 2]). Washout appearance was observed in 34/50 / 20/50 lesions = 68%/40% with CEUS and 31/50 / 31/50 lesions = 62%/62%) with MRI. Interobserver agreement was moderate for arterial hyperenhancement (ĸ = 0.511/0.565 [CEUS/MRI]) and "washout" (ĸ = 0.490/0.582 [CEUS/MRI]), fair for CEUS-LI-RADS category (ĸ = 0.309) and substantial for MRI-LI-RADS category (ĸ = 0.609). Intermodality agreement was fair for arterial hyperenhancement (ĸ = 0.329), slight to fair for "washout" (ĸ = 0.202) and LI-RADS category (ĸ = 0.218) CONCLUSION: Interobserver agreement is substantial for MRI-LI-RADS and only fair for CEUS-LI-RADS. This is mostly because interobserver agreement in the perception of washout appearance is better in MRI than in CEUS. Further refinement of the LI-RADS algorithms and increasing education and practice may be necessary to improve the concordance between CEUS and MRI for the final LI-RADS categorization. • CEUS-LI-RADS and MRI-LIRADS enable standardized non-invasive diagnosis of HCC in

  19. Structure of the human DNA-repair protein RAD52 containing surface mutations.

    PubMed

    Saotome, Mika; Saito, Kengo; Onodera, Keiichi; Kurumizaka, Hitoshi; Kagawa, Wataru

    2016-08-01

    The Rad52 protein is a eukaryotic single-strand DNA-annealing protein that is involved in the homologous recombinational repair of DNA double-strand breaks. The isolated N-terminal half of the human RAD52 protein (RAD52(1-212)) forms an undecameric ring structure with a surface that is mostly positively charged. In the present study, it was found that RAD52(1-212) containing alanine mutations of the charged surface residues (Lys102, Lys133 and Glu202) is highly amenable to crystallization. The structure of the mutant RAD52(1-212) was solved at 2.4 Å resolution. The structure revealed an association between the symmetry-related RAD52(1-212) rings, in which a partially unfolded, C-terminal region of RAD52 extended into the DNA-binding groove of the neighbouring ring in the crystal. The alanine mutations probably reduced the surface entropy of the RAD52(1-212) ring and stabilized the ring-ring association observed in the crystal.

  20. Comparison of Danish dichotomous and BI-RADS classifications of mammographic density.

    PubMed

    Hodge, Rebecca; Hellmann, Sophie Sell; von Euler-Chelpin, My; Vejborg, Ilse; Andersen, Zorana Jovanovic

    2014-06-01

    In the Copenhagen mammography screening program from 1991 to 2001, mammographic density was classified either as fatty or mixed/dense. This dichotomous mammographic density classification system is unique internationally, and has not been validated before. To compare the Danish dichotomous mammographic density classification system from 1991 to 2001 with the density BI-RADS classifications, in an attempt to validate the Danish classification system. The study sample consisted of 120 mammograms taken in Copenhagen in 1991-2001, which tested false positive, and which were in 2012 re-assessed and classified according to the BI-RADS classification system. We calculated inter-rater agreement between the Danish dichotomous mammographic classification as fatty or mixed/dense and the four-level BI-RADS classification by the linear weighted Kappa statistic. Of the 120 women, 32 (26.7%) were classified as having fatty and 88 (73.3%) as mixed/dense mammographic density, according to Danish dichotomous classification. According to BI-RADS density classification, 12 (10.0%) women were classified as having predominantly fatty (BI-RADS code 1), 46 (38.3%) as having scattered fibroglandular (BI-RADS code 2), 57 (47.5%) as having heterogeneously dense (BI-RADS 3), and five (4.2%) as having extremely dense (BI-RADS code 4) mammographic density. The inter-rater variability assessed by weighted kappa statistic showed a substantial agreement (0.75). The dichotomous mammographic density classification system utilized in early years of Copenhagen's mammographic screening program (1991-2001) agreed well with the BI-RADS density classification system.

  1. Impact of physicochemical parameters on in vitro assembly and disassembly kinetics of recombinant triple-layered rotavirus-like particles.

    PubMed

    Mellado, Maria Candida M; Mena, Jimmy A; Lopes, António; Ramírez, Octavio T; Carrondo, Manuel J T; Palomares, Laura A; Alves, Paula M

    2009-11-01

    Virus-like particles constitute potentially relevant vaccine candidates. Nevertheless, their behavior in vitro and assembly process needs to be understood in order to improve their yield and quality. In this study we aimed at addressing these issues and for that purpose triple- and double-layered rotavirus-like particles (TLP 2/6/7 and DLP 2/6, respectively) size and zeta potential were measured using dynamic light scattering at different physicochemical conditions, namely pH, ionic strength, and temperature. Both TLP and DLP were stable within a pH range of 3-7 and at 5-25 degrees C. Aggregation occurred at 35-45 degrees C and their disassembly became evident at 65 degrees C. The isoelectric points of TLP and DLP were 3.0 and 3.8, respectively. In vitro kinetics of TLP disassembly was monitored. Ionic strength, temperature, and the chelating agent employed determined disassembly kinetics. Glycerol (10%) stabilized TLP by preventing its disassembly. Disassembled TLP was able to reassemble by dialysis at high calcium conditions. VP7 monomers were added to DLP in the presence of calcium to follow in vitro TLP assembly kinetics; its assembly rate being mostly affected by pH. Finally, DLP and TLP were found to coexist under certain conditions as determined from all reaction products analyzed by capillary electrophoresis. Overall, these results contribute to the design of new strategies for the improvement of TLP yield and quality by reducing the VP7 detachment from TLP. Copyright 2009 Wiley Periodicals, Inc.

  2. Easily disassembled electrical connector for high voltage, high frequency connections

    DOEpatents

    Milner, Joseph R.

    1994-01-01

    An easily accessible electrical connector capable of rapid assembly and disassembly wherein a wide metal conductor sheet may be evenly contacted over the entire width of the conductor sheet by opposing surfaces on the connector which provide an even clamping pressure against opposite surfaces of the metal conductor sheet using a single threaded actuating screw.

  3. Montmorillonite-induced Bacteriophage φ6 Disassembly

    NASA Astrophysics Data System (ADS)

    Trusiak, A.; Gottlieb, P.; Katz, A.; Alimova, A.; Steiner, J. C.; Block, K. A.

    2012-12-01

    It is estimated that there are 1031 virus particles on Earth making viruses an order of magnitude more prevalent in number than prokaryotes with the vast majority of viruses being bacteriophages. Clays are a major component of soils and aquatic sediments and can react with RNA, proteins and bacterial biofilms. The clays in soils serve as an important moderator between phage and their host bacteria, helping to preserve the evolutionary balance. Studies on the effects of clays on viral infectivity have given somewhat contradictory results; possibly a consequence of clay-virus interactions being dependent on the unique structure of particular viruses. In this work, the interaction between montmorillonite and the bacteriophage φ6 is investigated. φ6 is a member of the cystovirus family that infects Pseudomonas syringe, a common plant pathogen. As a member of the cystovirus family with an enveloped structure, φ6 serves as a model for reoviruses, a human pathogen. Experiments were conducted with φ6 suspended in dilute, purified homoionic commercial-grade montmorillonite over a range of virus:clay ratios. At a 1:100000 virus:clay ratio, the clay reduced viral infectivity by 99%. The minimum clay to virus ratio which results in a measurable reduction of P. syringae infection is 1:1. Electron microscopy demonstrates that mixed suspensions of smectite and virus co-aggregate to form flocs encompassing virions within the smectite. Both free viral particles as well as those imbedded in the flocs are seen in the micrographs to be missing the envelope- leaving only the nucleocapsid (NC) intact; indicating that smectite inactivates the virus by envelope disassembly. These results have strong implications in the evolution of both the φ6 virus and its P. syringae host cells. TEM of aggregate showing several disassembled NCs.

  4. Replication-Dependent Sister Chromatid Recombination in Rad1 Mutants of Saccharomyces Cerevisiae

    PubMed Central

    Kadyk, L. C.; Hartwell, L. H.

    1993-01-01

    Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G(1) or the G(2) phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G(1), this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G(2) phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G(2). Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination. PMID:8454200

  5. Towards a Global Operational Altimeter Service: RADS

    NASA Astrophysics Data System (ADS)

    Naeije, M.; Schrama, E.; Mathers, L.; Scharroo, R.

    2001-12-01

    DEOS' anticipation of the need for global altimeter services started the Radar Altimeter Database System (RADS) project. Embedded in the Netherlands Earth Observation NETwork (NEONET), this project is supported by the Dutch government. After defining the database content, collecting altimeter and ancillary data from all available altimeter missions and combining them with the latest (correction) models, we have arrived at an (inter)nationally appreciated validated, calibrated and consistent altimeter data set, comprising over 15 years of valuable sea level, wave height and wind data. Whenever new data or knowledge arrives the database is updated. Major assets of RADS are the upgraded ERS orbits and the flexible data organization. This paper presents an overview of the work involved in establishing RADS: the I/O, enhancements, screening, formatting, harmonization, and CAL/VAL. The aim is to improve the algorithms for converting satellite data to the final geophysical products. Global altimeter data from various satellites are inter-compared or compared to external data, like tide gauges, wind speed measurements, etc. This has been used to establish the data's quality and to enhance algorithms for deriving the geophysical parameters. Also: ironing out inconsistencies in significant wave height, sea state, inverse barometer, wet troposphere corrections, orbits, biases, drifts, and time tagging. Access to the database at level~1 level is provided for by a web portal (\\tt http://www.deos.tudelft.nl/altim/rads). Here also status, higher level products, software, and literature can be obtained. Finally, examples are given of putting in RADS in research and education. We fully automated the Gulf Stream and El Niño web pages: Hovmuller diagrams and eddy kinetic energy plots are refreshed regularly. Furthermore, RADS has been successfully used at Delft Hydraulics in a data assimilation scheme for improving tides and storm surge predictions, showing the importance of near

  6. RadMap Installation Instructions

    EPA Pesticide Factsheets

    RadMap is an interactive desktop tool featuring a nationwide geographic information systems (GIS) map of long-term radiation monitoring locations across the United States with access to key information about the monitor and the area surrounding it.

  7. Casein kinase 2 promotes interaction between Rad17 and the 9-1-1 complex through constitutive phosphorylation of the C-terminal tail of human Rad17.

    PubMed

    Fukumoto, Yasunori; Takahashi, Kazuaki; Suzuki, Noriyuki; Ogra, Yasumitsu; Nakayama, Yuji; Yamaguchi, Naoto

    2018-06-15

    An interaction between the Rad17-RFC2-5 and 9-1-1 complexes is essential for ATR-Chk1 signaling, which is one of the major DNA damage checkpoints. Recently, we showed that the polyanionic C-terminal tail of human Rad17 and the embedded conserved sequence iVERGE are important for the interaction with 9-1-1 complex. Here, we show that Rad17-S667 in the C-terminal tail is constitutively phosphorylated in vivo in a casein kinase 2-dependent manner, and the phosphorylation is important for 9-1-1 interaction. The serine phosphorylation of Rad17 could be seen in the absence of exogenous genotoxic stress, and was mostly abolished by S667A substitution. Rad17-S667 was also phosphorylated when the C-terminal tail was fused with EGFP, but the phosphorylation was inhibited by two casein kinase 2 inhibitors. Furthermore, interaction between Rad17 and the 9-1-1 complex was inhibited by the casein kinase 2 inhibitor CX-4945/Silmitasertib, and the effect was dependent on the Rad17-S667 residue, indicating that S667 phosphorylation is the only role of casein kinase 2 in the 9-1-1 interaction. Our data raise the possibility that the C-terminal tail of vertebrate Rad17 regulates ATR-Chk1 signaling through multi-site phosphorylation in the iVERGE. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Actin filament curvature biases branching direction

    NASA Astrophysics Data System (ADS)

    Wang, Evan; Risca, Viviana; Chaudhuri, Ovijit; Chia, Jia-Jun; Geissler, Phillip; Fletcher, Daniel

    2012-02-01

    Actin filaments are key components of the cellular machinery, vital for a wide range of processes ranging from cell motility to endocytosis. Actin filaments can branch, and essential in this process is a protein complex known as the Arp2/3 complex, which nucleate new ``daughter'' filaments from pre-existing ``mother'' filaments by attaching itself to the mother filament. Though much progress has been made in understanding the Arp2/3-actin junction, some very interesting questions remain. In particular, F-actin is a dynamic polymer that undergoes a wide range of fluctuations. Prior studies of the Arp2/3-actin junction provides a very static notion of Arp2/3 binding. The question we ask is how differently does the Arp2/3 complex interact with a straight filament compared to a bent filament? In this study, we used Monte Carlo simulations of a surface-tethered worm-like chain to explore possible mechanisms underlying the experimental observation that there exists preferential branch formation by the Arp2/3 complex on the convex face of a curved filament. We show that a fluctuation gating model in which Arp2/3 binding to the actin filament is dependent upon a rare high-local-curvature shape fluctuation of the filament is consistent with the experimental data.

  9. Easily disassembled electrical connector for high voltage, high frequency connections

    DOEpatents

    Milner, J.R.

    1994-05-10

    An easily accessible electrical connector capable of rapid assembly and disassembly is described wherein a wide metal conductor sheet may be evenly contacted over the entire width of the conductor sheet by opposing surfaces on the connector which provide an even clamping pressure against opposite surfaces of the metal conductor sheet using a single threaded actuating screw. 13 figures.

  10. SYMPATHETIC SOLAR FILAMENT ERUPTIONS

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

    Wang, Rui; Liu, Ying D.; Zimovets, Ivan

    2016-08-10

    The 2015 March 15 coronal mass ejection as one of the two that together drove the largest geomagnetic storm of solar cycle 24 so far was associated with sympathetic filament eruptions. We investigate the relations between the different filaments involved in the eruption. A surge-like small-scale filament motion is confirmed as the trigger that initiated the erupting filament with multi-wavelength observations and using a forced magnetic field extrapolation method. When the erupting filament moved to an open magnetic field region, it experienced an obvious acceleration process and was accompanied by a C-class flare and the rise of another larger filamentmore » that eventually failed to erupt. We measure the decay index of the background magnetic field, which presents a critical height of 118 Mm. Combining with a potential field source surface extrapolation method, we analyze the distributions of the large-scale magnetic field, which indicates that the open magnetic field region may provide a favorable condition for F2 rapid acceleration and have some relation with the largest solar storm. The comparison between the successful and failed filament eruptions suggests that the confining magnetic field plays an important role in the preconditions for an eruption.« less

  11. Asymmetric ring structure of Vps4 required for ESCRT-III disassembly

    NASA Astrophysics Data System (ADS)

    Caillat, Christophe; Macheboeuf, Pauline; Wu, Yuanfei; McCarthy, Andrew A.; Boeri-Erba, Elisabetta; Effantin, Gregory; Göttlinger, Heinrich G.; Weissenhorn, Winfried; Renesto, Patricia

    2015-12-01

    The vacuolar protein sorting 4 AAA-ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here we present a 3.6-Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP-binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high-affinity and five low-affinity binding sites in vitro, consistent with conformational asymmetry induced on ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly.

  12. Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment.

    PubMed

    Eyboulet, Fanny; Cibot, Camille; Eychenne, Thomas; Neil, Helen; Alibert, Olivier; Werner, Michel; Soutourina, Julie

    2013-12-01

    Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3' endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes.

  13. A penny-shaped crack in a filament-reinforced matrix. I - The filament model. II - The crack problem

    NASA Technical Reports Server (NTRS)

    Erdogan, F.; Pacella, A. H.

    1974-01-01

    The study deals with the elastostatic problem of a penny-shaped crack in an elastic matrix which is reinforced by filaments or fibers perpendicular to the plane of the crack. An elastic filament model is first developed, followed by consideration of the application of the model to the penny-shaped crack problem in which the filaments of finite length are asymmetrically distributed around the crack. Since the primary interest is in the application of the results to studies relating to the fracture of fiber or filament-reinforced composites and reinforced concrete, the main emphasis of the study is on the evaluation of the stress intensity factor along the periphery of the crack, the stresses in the filaments or fibers, and the interface shear between the matrix and the filaments or fibers. Using the filament model developed, the elastostatic interaction problem between a penny-shaped crack and a slender inclusion or filament in an elastic matrix is formulated.

  14. CAD-RADS - a new clinical decision support tool for coronary computed tomography angiography.

    PubMed

    Foldyna, Borek; Szilveszter, Bálint; Scholtz, Jan-Erik; Banerji, Dahlia; Maurovich-Horvat, Pál; Hoffmann, Udo

    2018-04-01

    Coronary computed tomography angiography (CTA) has been established as an accurate method to non-invasively assess coronary artery disease (CAD). The proposed 'Coronary Artery Disease Reporting and Data System' (CAD-RADS) may enable standardised reporting of the broad spectrum of coronary CTA findings related to the presence, extent and composition of coronary atherosclerosis. The CAD-RADS classification is a comprehensive tool for summarising findings on a per-patient-basis dependent on the highest-grade coronary artery lesion, ranging from CAD-RADS 0 (absence of CAD) to CAD-RADS 5 (total occlusion of a coronary artery). In addition, it provides suggestions for clinical management for each classification, including further testing and therapeutic options. Despite some limitations, CAD-RADS may facilitate improved communication between imagers and patient caregivers. As such, CAD-RADS may enable a more efficient use of coronary CTA leading to more accurate utilisation of invasive coronary angiograms. Furthermore, widespread use of CAD-RADS may facilitate registry-based research of diagnostic and prognostic aspects of CTA. • CAD-RADS is a tool for standardising coronary CTA reports. • CAD-RADS includes clinical treatment recommendations based on CTA findings. • CAD-RADS has the potential to reduce variability of CTA reports.

  15. Tungsten Filament Fire

    ERIC Educational Resources Information Center

    Ruiz, Michael J.; Perkins, James

    2016-01-01

    We safely remove the outer glass bulb from an incandescent lamp and burn up the tungsten filament after the glass is removed. This demonstration dramatically illustrates the necessity of a vacuum or inert gas for the environment surrounding the tungsten filament inside the bulb. Our approach has added historical importance since the incandescent…

  16. RadNet Air Data From Salt Lake City, UT

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for Salt Lake City, UT from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  17. RadNet Air Data From New York City, NY

    EPA Pesticide Factsheets

    This page presents radiation air monitoring and air filter analysis data for New York City, NY from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

  18. Electron emitting filaments for electron discharge devices

    DOEpatents

    Leung, Ka-Ngo; Pincosy, Philip A.; Ehlers, Kenneth W.

    1988-01-01

    Electrons are copiously emitted by a device comprising a loop-shaped filament made of lanthanum hexaboride. The filament is directly heated by an electrical current produced along the filament by a power supply connected to the terminal legs of the filament. To produce a filament, a diamond saw or the like is used to cut a slice from a bar made of lanthanum hexaboride. The diamond saw is then used to cut the slice into the shape of a loop which may be generally rectangular, U-shaped, hairpin-shaped, zigzag-shaped, or generally circular. The filaments provide high electron emission at a relatively low operating temperature, such as 1600.degree. C. To achieve uniform heating, the filament is formed with a cross section which is tapered between the opposite ends of the filament to compensate for non-uniform current distribution along the filament due to the emission of electrons from the filament.

  19. Electron emitting filaments for electron discharge devices

    DOEpatents

    Leung, K.N.; Pincosy, P.A.; Ehlers, K.W.

    1983-06-10

    Electrons are copiously emitted by a device comprising a loop-shaped filament made of lanthanum hexaboride. The filament is directly heated by an electrical current produced along the filament by a power supply connected to the terminal legs of the filament. To produce a filament, a diamond saw or the like is used to cut a slice from a bar made of lanthanum hexaboride. The diamond saw is then used to cut the slice into the shape of a loop which may be generally rectangular, U-shaped, hairpin-shaped, zigzag-shaped, or generally circular. The filaments provide high electron emission at a relatively low operating temperature, such as 1600/sup 0/C. To achieve uniform heating, the filament is formed with a cross section which is tapered between the opposite ends of the filament to compensate for nonuniform current distribution along the filament due to the emission of electrons from the filament.

  20. Variations in the sonographic measurement techniques of BI-RADS 3 breast masses.

    PubMed

    Francisco, Juliana; Jales, Rodrigo Menezes; de Oliveira, André Desuó Bueno; Arguello, Carlos Henrique Francisco; Derchain, Sophie

    2017-06-01

    To evaluate the differences in sonographic (US) distance and volume measurements from different sonologists and identify the optimal parameters to avoid clinically relevant variations in the measurement of BI-RADS 3 breast masses. For this cross-sectional study with prospectively collected data, four physicians with various levels of experience in US, herein called sonologists, performed distance and volume US measurements of 80 masses classified as BI-RADS 3. The Cochran Q test was used to compare the matched sets of rates of clinically relevant variability between all pairs of sonologists' measurements. There were clinically relevant differences between sonologists in the measurements of the longest diameter (range, 17.5-43.7%, p = 0.003), the longest diameter perpendicular to the previous one (anteroposterior diameter) (17.5-33.7%, p = 0.06), the third diameter orthogonal to the plane defined by the previous two (transverse diameter) (28.7-55%, p = 0.001), and at least two of those three diameters (18.7-38.7%, p = 0.015). The smallest clinically relevant differences were observed with volume measurements (range of differences, 6.2-13.7%, p = 0.51). Volume measurement technique was associated with the least variations, whereas distance measurements, which are used routinely, were associated with unacceptable rates of clinically relevant variations. © 2016 Wiley Periodicals, Inc. J Clin Ultrasound 45:252-260, 2017. © 2017 Wiley Periodicals, Inc.

  1. The domain organization of the bacterial intermediate filament-like protein crescentin is important for assembly and function

    PubMed Central

    Cabeen, Matthew T; Herrmann, Harald; Jacobs-Wagner, Christine

    2011-01-01

    Crescentin is a bacterial filament-forming protein that exhibits domain organization features found in metazoan intermediate filament (IF) proteins. Structure-function studies of eukaryotic IFs have been hindered by a lack of simple genetic systems and easily quantifiable phenotypes. Here we exploit the characteristic localization of the crescentin structure along the inner curvature of Caulobacter crescentus cells and the loss of cell curvature associated with impaired crescentin function to analyze the importance of the domain organization of crescentin. By combining biochemistry and ultrastructural analysis in vitro with cellular localization and functional studies, we show that crescentin requires its distinctive domain organization, and furthermore that different structural elements have distinct structural and functional contributions. The head domain can be functionally subdivided into two subdomains; the first (amino-terminal) is required for function but not assembly, while the second is necessary for structure assembly. The rod domain is similarly required for structure assembly, and the linker L1 appears important to prevent runaway assembly into nonfunctional aggregates. The data also suggest that the stutter and the tail domain have critical functional roles in stabilizing crescentin structures against disassembly by monovalent cations in the cytoplasm. This study suggests that the IF-like behavior of crescentin is a consequence of its domain organization, implying that the IF protein layout is an adaptable cytoskeletal motif, much like the actin and tubulin folds, that is broadly exploited for various functions throughout life from bacteria to humans. © 2011 Wiley-Liss, Inc. PMID:21360832

  2. Unwinding motion of a twisted active region filament

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

    Yan, X. L.; Xue, Z. K.; Kong, D. F.

    To better understand the structures of active region filaments and the eruption process, we study an active region filament eruption in active region NOAA 11082 in detail on 2010 June 22. Before the filament eruption, the opposite unidirectional material flows appeared in succession along the spine of the filament. The rising of the filament triggered two B-class flares at the upper part of the filament. As the bright material was injected into the filament from the sites of the flares, the filament exhibited a rapid uplift accompanying the counterclockwise rotation of the filament body. From the expansion of the filament,more » we can see that the filament consisted of twisted magnetic field lines. The total twist of the filament is at least 5π obtained by using a time slice method. According to the morphology change during the filament eruption, it is found that the active region filament was a twisted flux rope and its unwinding motion was like a solar tornado. We also find that there was a continuous magnetic helicity injection before and during the filament eruption. It is confirmed that magnetic helicity can be transferred from the photosphere to the filament. Using the extrapolated potential fields, the average decay index of the background magnetic fields over the filament is 0.91. Consequently, these findings imply that the mechanism of solar filament eruption could be due to the kink instability and magnetic helicity accumulation.« less

  3. Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment

    PubMed Central

    Eyboulet, Fanny; Cibot, Camille; Eychenne, Thomas; Neil, Helen; Alibert, Olivier; Werner, Michel; Soutourina, Julie

    2013-01-01

    Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3′ endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes. PMID:24298055

  4. A thyroid nodule classification method based on TI-RADS

    NASA Astrophysics Data System (ADS)

    Wang, Hao; Yang, Yang; Peng, Bo; Chen, Qin

    2017-07-01

    Thyroid Imaging Reporting and Data System(TI-RADS) is a valuable tool for differentiating the benign and the malignant thyroid nodules. In clinic, doctors can determine the extent of being benign or malignant in terms of different classes by using TI-RADS. Classification represents the degree of malignancy of thyroid nodules. TI-RADS as a classification standard can be used to guide the ultrasonic doctor to examine thyroid nodules more accurately and reliably. In this paper, we aim to classify the thyroid nodules with the help of TI-RADS. To this end, four ultrasound signs, i.e., cystic and solid, echo pattern, boundary feature and calcification of thyroid nodules are extracted and converted into feature vectors. Then semi-supervised fuzzy C-means ensemble (SS-FCME) model is applied to obtain the classification results. The experimental results demonstrate that the proposed method can help doctors diagnose the thyroid nodules effectively.

  5. Artificial biofilms establish the role of matrix interactions in staphylococcal biofilm assembly and disassembly.

    PubMed

    Stewart, Elizabeth J; Ganesan, Mahesh; Younger, John G; Solomon, Michael J

    2015-08-14

    We demonstrate that the microstructural and mechanical properties of bacterial biofilms can be created through colloidal self-assembly of cells and polymers, and thereby link the complex material properties of biofilms to well understood colloidal and polymeric behaviors. This finding is applied to soften and disassemble staphylococcal biofilms through pH changes. Bacterial biofilms are viscoelastic, structured communities of cells encapsulated in an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, and DNA. Although the identity and abundance of EPS macromolecules are known, how these matrix materials interact with themselves and bacterial cells to generate biofilm morphology and mechanics is not understood. Here, we find that the colloidal self-assembly of Staphylococcus epidermidis RP62A cells and polysaccharides into viscoelastic biofilms is driven by thermodynamic phase instability of EPS. pH conditions that induce phase instability of chitosan produce artificial S. epidermidis biofilms whose mechanics match natural S. epidermidis biofilms. Furthermore, pH-induced solubilization of the matrix triggers disassembly in both artificial and natural S. epidermidis biofilms. This pH-induced disassembly occurs in biofilms formed by five additional staphylococcal strains, including three clinical isolates. Our findings suggest that colloidal self-assembly of cells and matrix polymers produces biofilm viscoelasticity and that biofilm control strategies can exploit this mechanism.

  6. Artificial biofilms establish the role of matrix interactions in staphylococcal biofilm assembly and disassembly

    PubMed Central

    Stewart, Elizabeth J.; Ganesan, Mahesh; Younger, John G.; Solomon, Michael J.

    2015-01-01

    We demonstrate that the microstructural and mechanical properties of bacterial biofilms can be created through colloidal self-assembly of cells and polymers, and thereby link the complex material properties of biofilms to well understood colloidal and polymeric behaviors. This finding is applied to soften and disassemble staphylococcal biofilms through pH changes. Bacterial biofilms are viscoelastic, structured communities of cells encapsulated in an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, and DNA. Although the identity and abundance of EPS macromolecules are known, how these matrix materials interact with themselves and bacterial cells to generate biofilm morphology and mechanics is not understood. Here, we find that the colloidal self-assembly of Staphylococcus epidermidis RP62A cells and polysaccharides into viscoelastic biofilms is driven by thermodynamic phase instability of EPS. pH conditions that induce phase instability of chitosan produce artificial S. epidermidis biofilms whose mechanics match natural S. epidermidis biofilms. Furthermore, pH-induced solubilization of the matrix triggers disassembly in both artificial and natural S. epidermidis biofilms. This pH-induced disassembly occurs in biofilms formed by five additional staphylococcal strains, including three clinical isolates. Our findings suggest that colloidal self-assembly of cells and matrix polymers produces biofilm viscoelasticity and that biofilm control strategies can exploit this mechanism. PMID:26272750

  7. Cinnamon extract inhibits tau aggregation associated with Alzheimer’s Disease in vitro

    USDA-ARS?s Scientific Manuscript database

    An aqueous extract of Ceylon cinnamon (C. zeylanicum) was found to inhibit tau aggregation and filament formation, hallmarks of Alzheimer’s disease (AD) in vitro using brain cells taken from patients who died with AD. The extract also promoted complete disassembly of recombinant tau filaments, and ...

  8. Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells

    PubMed Central

    Kampourakis, Thomas; Yan, Ziqian; Gautel, Mathias; Sun, Yin-Biao; Irving, Malcolm

    2014-01-01

    Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility. PMID:25512492

  9. Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells.

    PubMed

    Kampourakis, Thomas; Yan, Ziqian; Gautel, Mathias; Sun, Yin-Biao; Irving, Malcolm

    2014-12-30

    Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.

  10. Role of a non-canonical surface of Rad6 in ubiquitin conjugating activity

    PubMed Central

    Kumar, Pankaj; Magala, Pearl; Geiger-Schuller, Kathryn R.; Majumdar, Ananya; Tolman, Joel R.; Wolberger, Cynthia

    2015-01-01

    Rad6 is a yeast E2 ubiquitin conjugating enzyme that monoubiquitinates histone H2B in conjunction with the E3, Bre1, but can non-specifically modify histones on its own. We determined the crystal structure of a Rad6∼Ub thioester mimic, which revealed a network of interactions in the crystal in which the ubiquitin in one conjugate contacts Rad6 in another. The region of Rad6 contacted is located on the distal face of Rad6 opposite the active site, but differs from the canonical E2 backside that mediates free ubiquitin binding and polyubiquitination activity in other E2 enzymes. We find that free ubiquitin interacts weakly with both non-canonical and canonical backside residues of Rad6 and that mutations of non-canonical residues have deleterious effects on Rad6 activity comparable to those observed to mutations in the canonical E2 backside. The effect of non-canonical backside mutations is similar in the presence and absence of Bre1, indicating that contacts with non-canonical backside residues govern the intrinsic activity of Rad6. Our findings shed light on the determinants of intrinsic Rad6 activity and reveal new ways in which contacts with an E2 backside can regulate ubiquitin conjugating activity. PMID:26286193

  11. Interdependence of the rad50 hook and globular domain functions.

    PubMed

    Hohl, Marcel; Kochańczyk, Tomasz; Tous, Cristina; Aguilera, Andrés; Krężel, Artur; Petrini, John H J

    2015-02-05

    Rad50 contains a conserved Zn(2+) coordination domain (the Rad50 hook) that functions as a homodimerization interface. Hook ablation phenocopies Rad50 deficiency in all respects. Here, we focused on rad50 mutations flanking the Zn(2+)-coordinating hook cysteines. These mutants impaired hook-mediated dimerization, but recombination between sister chromatids was largely unaffected. This may reflect that cohesin-mediated sister chromatid interactions are sufficient for double-strand break repair. However, Mre11 complex functions specified by the globular domain, including Tel1 (ATM) activation, nonhomologous end joining, and DNA double-strand break end resection were affected, suggesting that dimerization exerts a broad influence on Mre11 complex function. These phenotypes were suppressed by mutations within the coiled-coil and globular ATPase domains, suggesting a model in which conformational changes in the hook and globular domains are transmitted via the extended coils of Rad50. We propose that transmission of spatial information in this manner underlies the regulation of Mre11 complex functions. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Fabrication of Polylactide Nanocomposite Filament Using Melt Extrusion and Filament Characterization for 3D Printing

    NASA Astrophysics Data System (ADS)

    Jain, Shrenik Kumar

    Fused deposition modeling (FDM) technology uses thermoplastic filament for layer by layer fabrication of objects. To make functional objects with desired properties, composite filaments are required in the FDM. In this thesis, less expensive mesoporous Nano carbon (NC) and carbon nanotube (CNT) infused in Polylactide (PLA) thermoplastic filaments were fabricated to improve the electrical properties and maintain sufficient strength for 3D printing. Solution blending was used for nanocomposite fabrication and melt extrusion was employed to make cylindrical filaments. Mechanical and electrical properties of 1 to 20 wt% of NC and 1 to 3 wt% of CNT filaments were investigated and significant improvement of conductivity (3.76 S/m) and sufficient yield strength (35MPa) were obtained. Scanning electron microscopy (SEM) images exhibited uniform dispersion of nanoparticles in polymer matrix and differential scanning calorimetry (DSC) results showed no significant changes in the glass transition temperature (Tg) for all the compositions. Perspective uses of this filament are for fabrication of electrical wires in 3D printed robots, drones, prosthetics, orthotics and others.

  13. The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in Vitro*

    PubMed Central

    Haldeman, Brian D.; Brizendine, Richard K.; Facemyer, Kevin C.; Baker, Josh E.; Cremo, Christine R.

    2014-01-01

    Actin-myosin interactions are well studied using soluble myosin fragments, but little is known about effects of myosin filament structure on mechanochemistry. We stabilized unphosphorylated smooth muscle myosin (SMM) and phosphorylated smooth muscle myosin (pSMM) filaments against ATP-induced depolymerization using a cross-linker and attached fluorescent rhodamine (XL-Rh-SMM). Electron micrographs showed that these side polar filaments are very similar to unmodified filaments. They are ∼0.63 μm long and contain ∼176 molecules. Rate constants for ATP-induced dissociation and ADP release from acto-myosin for filaments and S1 heads were similar. Actin-activated ATPases of SMM and XL-Rh-SMM were similarly regulated. XL-Rh-pSMM filaments moved processively on F-actin that was bound to a PEG brush surface. ATP dependence of filament velocities was similar to that for solution ATPases at high [actin], suggesting that both processes are limited by the same kinetic step (weak to strong transition) and therefore are attachment-limited. This differs from actin sliding over myosin monomers, which is primarily detachment-limited. Fitting filament data to an attachment-limited model showed that approximately half of the heads are available to move the filament, consistent with a side polar structure. We suggest the low stiffness subfragment 2 (S2) domain remains unhindered during filament motion in our assay. Actin-bound negatively displaced heads will impart minimal drag force because of S2 buckling. Given the ADP release rate, the velocity, and the length of S2, these heads will detach from actin before slack is taken up into a backwardly displaced high stiffness position. This mechanism explains the lack of detachment-limited kinetics at physiological [ATP]. These findings address how nonlinear elasticity in assemblies of motors leads to efficient collective force generation. PMID:24907276

  14. Jab1 Mediates Protein Degradation of Rad9/Rad1/Hus1 Checkpoint Complex

    PubMed Central

    Huang, Jin; Yuan, Honglin; Lu, Chongyuan; Liu, Ximeng; Cao, Xu; Wan, Mei

    2009-01-01

    Summary The Rad1-Rad9-Hus1 (9-1-1) complex serves a dual role as a DNA-damage sensor in checkpoint signaling and as a mediator in DNA repair pathway. However, the intercellular mechanisms that regulate 9-1-1 complex are poorly understood. Jab1, the fifth component of the COP9 signalosome complex, plays a central role in the degradation of multiple proteins and is emerging as an important regulator in cancer development. Here, we tested the hypothesis that Jab1 controls the protein stability of the 9-1-1 complex via the proteosome pathway. We provide evidence that Jab1 physically associates with the 9-1-1 complex. This association is mediated through direct interaction between Jab1 and Rad1, one of the subunits of 9-1-1 complex. Importantly, Jab1 causes the translocation of the 9-1-1 complex from the nucleus to the cytoplasm, mediating rapid degradation of the 9-1-1 complex via 26S proteasome. Furthermore, Jab1 significantly suppresses checkpoint signaling activation, DNA synthesis recovery from blockage and cell viability after replication stresses such as UV exposure, γ radiation and hydroxyurea treatment. These results suggest that Jab1 is an important regulator for 9-1-1 protein stability control in cells, which may provide novel information on the involvement of Jab1 in checkpoint and DNA repair signaling in response to DNA damage. PMID:17583730

  15. Molecular disassembly of rice and lotus starches during thermal processing and its effect on starch digestibility.

    PubMed

    Wang, Shujun; Sun, Yue; Wang, Jinrong; Wang, Shuo; Copeland, Les

    2016-02-01

    The molecular disassembly of starch during thermal processing is a major determinant for the susceptibility of starch to enzymatic digestion. In the present study, the effects of thermal processing on the disassembly of the granular structure and the in vitro enzymatic digestibility of rice and lotus starches were investigated. After heating at 50 °C, rice and lotus starches did not show significant changes in granular morphology, long-range crystallinity and short-range molecular order. As the temperature increased to 60 °C, rice starch underwent a partial gelatinization followed by an incomplete disruption of granular morphology, crystallites and molecular order. In contrast, lotus starch was almost completely gelatinized at 60 °C. At 70 °C or higher, both starches were fully gelatinized with complete disruption of the micro and macro structures. Our results show that gelatinization greatly increased the in vitro enzymatic digestibility of both starches, but that the degree of disassembly of the starch structure during thermal processing was not a major determinant of the digestibility of gelatinized starch.

  16. Aberrant intermediate filament and synaptophysin expression is a frequent event in malignant melanoma: an immunohistochemical study of 73 cases.

    PubMed

    Romano, Ryan C; Carter, Jodi M; Folpe, Andrew L

    2015-08-01

    Malignant melanomas are known to express vimentin, among other intermediate filaments. Though anomalous keratin expression by malignant melanoma has been reported, its frequency is not well-established and this phenomenon is not well-known. We have seen in consultation a number of malignant melanomas with anomalous expression of keratin, other intermediate filaments, or synaptophysin, and therefore studied a large group of primary and metastatic melanomas to determine the frequency of these events. About 73 cases of malignant melanoma (22 primaries and 51 metastases) from 71 patients (51 male, 20 female; mean 59 years, range 17-87 years) were retrieved from our archives. Prior diagnoses were confirmed by re-review of hematoxylin and eosin sections and relevant (e.g., S100 protein, HMB45, Melan-A, and tyrosinase) immunohistochemical studies. Available sections were immunostained for keratin (OSCAR and AE1/AE3 antibodies), desmin, neurofilament protein, glial fibrillary acidic protein, synaptophysin, and chromogranin A. Not all cases could be tested for all markers. Cases were predominantly epithelioid (48/73, 66%) or spindle cell/desmoplastic (25/73, 34%). S100 protein, Melan-A, HMB45, and tyrosinase were positive in 60/65 (92%), 34/64 (53%), 30/60 (50%), 25/48 (52%) of cases, respectively. All five S100-protein-negative cases expressed at least one of the other melanocytic markers: Melan-A (two of four, 50%), HMB45 (two of three, 67%), and tyrosinase (one of two, 50%). All cases expressed at least one melanocytic marker. Cases were positive for keratin (OSCAR, 17/61, 28%; AE1/AE3, 16/40, 40%), desmin (11/47, 24%), neurofilament protein (5/31, 16%), glial fibrillary acidic protein (3/32, 9%), and synaptophysin (10/34, 29%), typically only in a minority of cells. Chromogranin was negative (0/32, 0%). Altogether 9/73 cases (12%) showed expression of >1 intermediate filament. All S100-protein-negative melanomas showed anomalous intermediate filament expression (keratin

  17. Synthesis of Zeolites Using the ADOR (Assembly-Disassembly-Organization-Reassembly) Route

    PubMed Central

    Wheatley, Paul S.; Čejka, Jiří; Morris, Russell E.

    2016-01-01

    Zeolites are an important class of materials that have wide ranging applications such as heterogeneous catalysts and adsorbents which are dependent on their framework topology. For new applications or improvements to existing ones, new zeolites with novel pore systems are desirable. We demonstrate a method for the synthesis of novel zeolites using the ADOR route. ADOR is an acronym for Assembly, Disassembly, Organization and Reassembly. This synthetic route takes advantage of the assembly of a relatively poorly stable that which can be selectively disassembled into a layered material. The resulting layered intermediate can then be organized in different manners by careful chemical manipulation and then reassembled into zeolites with new topologies. By carefully controlling the organization step of the synthetic pathway, new zeolites with never before seen topologies are capable of being synthesized. The structures of these new zeolites are confirmed using powder X-ray diffraction and further characterized by nitrogen adsorption and scanning electron microscopy. This new synthetic pathway for zeolites demonstrates its capability to produce novel frameworks that have never been prepared by traditional zeolite synthesis techniques. PMID:27078165

  18. Synthesis of Zeolites Using the ADOR (Assembly-Disassembly-Organization-Reassembly) Route.

    PubMed

    Wheatley, Paul S; Čejka, Jiří; Morris, Russell E

    2016-04-03

    Zeolites are an important class of materials that have wide ranging applications such as heterogeneous catalysts and adsorbents which are dependent on their framework topology. For new applications or improvements to existing ones, new zeolites with novel pore systems are desirable. We demonstrate a method for the synthesis of novel zeolites using the ADOR route. ADOR is an acronym for Assembly, Disassembly, Organization and Reassembly. This synthetic route takes advantage of the assembly of a relatively poorly stable that which can be selectively disassembled into a layered material. The resulting layered intermediate can then be organized in different manners by careful chemical manipulation and then reassembled into zeolites with new topologies. By carefully controlling the organization step of the synthetic pathway, new zeolites with never before seen topologies are capable of being synthesized. The structures of these new zeolites are confirmed using powder X-ray diffraction and further characterized by nitrogen adsorption and scanning electron microscopy. This new synthetic pathway for zeolites demonstrates its capability to produce novel frameworks that have never been prepared by traditional zeolite synthesis techniques.

  19. Evidence that Hsc70 Is Associated with Cucumber Necrosis Virus Particles and Plays a Role in Particle Disassembly

    PubMed Central

    Alam, Syed Benazir

    2016-01-01

    ABSTRACT Uncoating of a virus particle to expose its nucleic acid is a critical aspect of the viral multiplication cycle, as it is essential for the establishment of infection. In the present study, we investigated the role of plant HSP70 homologs in the uncoating process of Cucumber necrosis virus (CNV), a nonenveloped positive-sense single-stranded RNA [(+)ssRNA] virus having a T=3 icosahedral capsid. We have found through Western blot analysis and mass spectrometry that the HSP70 homolog Hsc70-2 copurifies with CNV particles. Virus overlay and immunogold labeling assays suggest that Hsc70-2 is physically bound to virions. Furthermore, trypsin digestion profiles suggest that the bound Hsc70-2 is partially protected by the virus, indicating an intimate association with particles. In investigating a possible role of Hsc70-2 in particle disassembly, we showed that particles incubated with Hsp70/Hsc70 antibody produce fewer local lesions than those incubated with prebleed control antibody on Chenopodium quinoa. In conjunction, CNV virions purified using CsCl and having undetectable amounts of Hsc70-2 produce fewer local lesions. We also have found that plants with elevated levels of HSP70/Hsc70 produce higher numbers of local lesions following CNV inoculation. Finally, incubation of recombinant Nicotiana benthamiana Hsc70-2 with virus particles in vitro leads to conformational changes or partial disassembly of capsids as determined by transmission electron microscopy, and particles are more sensitive to chymotrypsin digestion. This is the first report suggesting that a cellular Hsc70 chaperone is involved in disassembly of a plant virus. IMPORTANCE Virus particles must disassemble and release their nucleic acid in order to establish infection in a cell. Despite the importance of disassembly in the ability of a virus to infect its host, little is known about this process, especially in the case of nonenveloped spherical RNA viruses. Previous work has shown that host

  20. ChemRad Sample Form Instructions

    EPA Pesticide Factsheets

    These instructons are intended to assist registered users of the EPA Region 8 Drinking Water Watch website who would like to create ChemRad Sample Forms for monitoring that is required during the current year.

  1. The Reach Address Database (RAD)

    EPA Pesticide Factsheets

    The Reach Address Database (RAD) stores reach address information for each Water Program feature that has been linked to the underlying surface water features (streams, lakes, etc) in the National Hydrology Database (NHD) Plus dataset.

  2. In-service inspection of steam turbine blades without disassembly

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

    Reinhart, E.R.

    1987-01-01

    Loss of utility plant availability as a result of failure-causing cracks in steam turbine blades makes early detection of this problem critical. An Electric Power Research Institute survey, conducted as part of project RP 1266-24, indicated that 72% of turbine blade failures in fossil power plants occur in low-pressure (LP) turbines with half of all blade failures occurring in the last two blade stages (L-0 and L-1 rows). Failures are generally associated with blade tailing edges and root areas. Project RP 1266-24 also found that 79% of the blade problems in LP turbines were cracks. A turbine design of particularmore » concern has been the Westinghouse Building Block (B.B.) 73. Reinhart and Associates has successfully inspected seven in-place B.B. 73 units for six utilities during the past 3 yr, as well as several disassembled turbines of other manufacturers and designs. These examinations consisted of visual and eddy-current examinations of the blade roots and trailing edges. The in-place inspections were performed using prototype manipulation devices to gain access to the blades through the hand holes. The only disassembly required to gain access for the examinations was the removal of the man-way covers on the main shell and the hand-hole covers on the outer cylinder covering the L-0 and L-1 blade rows.« less

  3. Ultrastructural characteristics of tau filaments in tauopathies: immuno-electron microscopic demonstration of tau filaments in tauopathies.

    PubMed

    Arima, Kunimasa

    2006-10-01

    The microtubule-associated protein tau aggregates into filaments in the form of neurofibrillary tangles, neuropil threads and argyrophilic grains in neurons, in the form of variable astrocytic tangles in astrocytes and in the form of coiled bodies and argyrophilic threads in oligodendrocytes. These tau filaments may be classified into two types, straight filaments or tubules with 9-18 nm diameters and "twisted ribbons" composed of two parallel aligned components. In the same disease, the fine structure of tau filaments in glial cells roughly resembles that in neurons. In sporadic tauopathies, individual tau filaments show characteristic sizes, shapes and arrangements, and therefore contribute to neuropathologic differential diagnosis. In frontotemporal dementias caused by tau gene mutations, variable filamentous profiles were observed in association with mutation sites and insoluble tau isoforms, including straight filaments or tubules, paired helical filament-like filaments, and twisted ribbons. Pre-embedding immunoelectron microscopic studies were carried out using anti-3-repeat tau and anti-4-repeat tau specific antibodies, RD3 and RD4. Straight tubules in neuronal and astrocytic Pick bodies were immunolabeled by the anti-3-repeat tau antibody. The anti-4-repeat tau antibody recognized abnormal tubules comprising neurofibrillary tangles, coiled bodies and argyrophilic threads in progressive supranuclear palsy (PSP) and corticobasal degeneration. In the pre-embedding immunoelectron microscopic study using the phosphorylated tau AT8 antibody, tuft-shaped astrocytes of PSP were found to be composed of bundles of abnormal tubules in processes and perikarya of protoplasmic astrocytes. In this study, the 3-repeat tau or 4-repeat tau epitope was detected in situ at the ultrastructural level in abnormal tubules in representative pathological lesions in Pick's disease, PSP and corticobasal degeneration.

  4. Side-binding proteins modulate actin filament dynamics

    PubMed Central

    Crevenna, Alvaro H; Arciniega, Marcelino; Dupont, Aurélie; Mizuno, Naoko; Kowalska, Kaja; Lange, Oliver F; Wedlich-Söldner, Roland; Lamb, Don C

    2015-01-01

    Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of filaments to polymerize and depolymerize at their ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface. We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of flexibility as well as effects on fragmentation and growth suggest that the observed kinetic diversity arises from structural alteration. Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics. DOI: http://dx.doi.org/10.7554/eLife.04599.001 PMID:25706231

  5. Role of Intermediate Filaments in Vesicular Traffic.

    PubMed

    Margiotta, Azzurra; Bucci, Cecilia

    2016-04-25

    Intermediate filaments are an important component of the cellular cytoskeleton. The first established role attributed to intermediate filaments was the mechanical support to cells. However, it is now clear that intermediate filaments have many different roles affecting a variety of other biological functions, such as the organization of microtubules and microfilaments, the regulation of nuclear structure and activity, the control of cell cycle and the regulation of signal transduction pathways. Furthermore, a number of intermediate filament proteins have been involved in the acquisition of tumorigenic properties. Over the last years, a strong involvement of intermediate filament proteins in the regulation of several aspects of intracellular trafficking has strongly emerged. Here, we review the functions of intermediate filaments proteins focusing mainly on the recent knowledge gained from the discovery that intermediate filaments associate with key proteins of the vesicular membrane transport machinery. In particular, we analyze the current understanding of the contribution of intermediate filaments to the endocytic pathway.

  6. The role of different PI-RADS versions in prostate multiparametric magnetic resonance tomography assessment.

    PubMed

    Aliukonis, Paulius; Letauta, Tadas; Briedienė, Rūta; Naruševičiūtė, Ieva; Letautienė, Simona

    2017-01-01

    Background . Standardised Prostate Imaging Reporting and Data System (PI-RADS) guidelines for the assessment of prostate alterations were designed for the assessment of prostate pathology. Published by the ESUR in 2012, PI-RADS v1 was based on the total score of different MRI sequences with subsequent calculation. PI-RADS v2 was published by the American College of Radiology in 2015 and featured different assessment criteria for prostate peripheral and transitory zones. Aim . To assess the correlations of PI-RADS v1 and PI-RADS v2 with Gleason score values and to define their predictive values of the diagnosis of prostate cancer. Materials and methods . A retrospective analysis of 66 patients. Prostate specific antigen (PSA) value and the Gleason score (GS) were assessed. One the most malignant focal lesion was selected in the peripheral zone of each lobe of the prostate (91 in total). Statistical analysis was carried out applying SPSS software, v.23, p < 0.05. Results . Focal lesions assessed by PI-RADS v1 score: 10% - 1, 12% - 2, 41% - 3, 23% - 4, 14% - 5. Assessment applying PI-RADS v.2: 20% - 1, 7.5% - 2, 26%, 29.5%, and 17% were assessed by 3, 4, and 5 scores. Statistically relevant correlation was found only between GS and PI-RADS ( p = 0.033). The positive predictive value of both versions of PI-RADS - 75%, negative predictive value of PI-RADS v1 - 46%, PI-RADS v2 - 43%. Conclusions . PI-RADS v1 was more statistically relevant in assessing the grade of tumour. Prediction values were similar in both versions.

  7. RNF168 forms a functional complex with RAD6 during the DNA damage response

    PubMed Central

    Liu, Chao; Wang, Degui; Wu, Jiaxue; Keller, Jennifer; Ma, Teng; Yu, Xiaochun

    2013-01-01

    Summary Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, E2 ubiquitin conjugating enzymes are crucial for catalyzing substrate ubiquitination that recruits downstream DNA repair factors to DNA lesions. To identify novel E2 conjugating enzymes important for initiating the DNA-damage-induced ubiquitination cascade, we screened most of the known E2 enzymes and found that RAD6A and RAD6B function together with RNF168 in the ionizing radiation (IR)-induced DNA damage response. Similarly to RNF168-deficient cells, RAD6A- or RAD6B-deficient cells exhibit a reduction in DNA-damage-induced protein ubiquitination. Correspondingly, DNA-damage-induced foci formation of DNA damage repair proteins, such as BRCA1 and 53BP1, is impaired in the absence of RAD6A or RAD6B. Moreover, the RNF168–RAD6 complex targeted histone H1.2 for ubiquitination in vitro and regulated DNA-damage-induced histone H1.2 ubiquitination in vivo. Collectively, these data demonstrate that RNF168, in complex with RAD6A or RAD6B, is activated in the DNA-damage-induced protein ubiquitination cascade. PMID:23525009

  8. Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

    NASA Astrophysics Data System (ADS)

    Chen, Xuejing; Velmurugu, Yogambigai; Zheng, Guanqun; Park, Beomseok; Shim, Yoonjung; Kim, Youngchang; Liu, Lili; van Houten, Bennett; He, Chuan; Ansari, Anjum; Min, Jung-Hyun

    2015-01-01

    The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.

  9. Boolean gates on actin filaments

    NASA Astrophysics Data System (ADS)

    Siccardi, Stefano; Tuszynski, Jack A.; Adamatzky, Andrew

    2016-01-01

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications.

  10. Solid friction between soft filaments

    NASA Astrophysics Data System (ADS)

    Ward, Andrew; Hilitski, Feodor; Schwenger, Walter; Welch, David; Lau, A. W. C.; Vitelli, Vincenzo; Mahadevan, L.; Dogic, Zvonimir

    2015-06-01

    Any macroscopic deformation of a filamentous bundle is necessarily accompanied by local sliding and/or stretching of the constituent filaments. Yet the nature of the sliding friction between two aligned filaments interacting through multiple contacts remains largely unexplored. Here, by directly measuring the sliding forces between two bundled F-actin filaments, we show that these frictional forces are unexpectedly large, scale logarithmically with sliding velocity as in solid-like friction, and exhibit complex dependence on the filaments’ overlap length. We also show that a reduction of the frictional force by orders of magnitude, associated with a transition from solid-like friction to Stokes’s drag, can be induced by coating F-actin with polymeric brushes. Furthermore, we observe similar transitions in filamentous microtubules and bacterial flagella. Our findings demonstrate how altering a filament’s elasticity, structure and interactions can be used to engineer interfilament friction and thus tune the properties of fibrous composite materials.

  11. Solid friction between soft filaments

    DOE PAGES

    Ward, Andrew; Hilitski, Feodor; Schwenger, Walter; ...

    2015-03-02

    Any macroscopic deformation of a filamentous bundle is necessarily accompanied by local sliding and/or stretching of the constituent filaments. Yet the nature of the sliding friction between two aligned filaments interacting through multiple contacts remains largely unexplored. Here, by directly measuring the sliding forces between two bundled F-actin filaments, we show that these frictional forces are unexpectedly large, scale logarithmically with sliding velocity as in solid-like friction, and exhibit complex dependence on the filaments’ overlap length. We also show that a reduction of the frictional force by orders of magnitude, associated with a transition from solid-like friction to Stokes’s drag,more » can be induced by coating F-actin with polymeric brushes. Furthermore, we observe similar transitions in filamentous microtubules and bacterial flagella. In conclusion, our findings demonstrate how altering a filament’s elasticity, structure and interactions can be used to engineer interfilament friction and thus tune the properties of fibrous composite materials.« less

  12. Recent observations of the formation of filaments

    NASA Technical Reports Server (NTRS)

    Martin, Sara F.

    1986-01-01

    Two examples of the formation of small filaments in H alpha are described and illustrated. In both cases, the formation is seen to be the spontaneous appearance of strands of absorbing mass that evolve from no previous structure. The initial development of the filaments appears to consist of the accumulation of these absorptive strands along approximately parallel paths in a channel between large-scale, opposite polarity magnetic fields on either side of the filaments. The strands exhibit continuous changes in shape and degree of absorption which can be due to successive condensations resulting in new strands, mass motions within the strands, and outflow of the mass from the strands. For at least several hours before the formation of both filaments, small-scale fragments of opposite polarity, line-of-sight magnetic flux adjacent to or immediately below the filaments, and at the ends of the filaments, were cancelling. This type of magnetic flux disappearance continued during the development of the filaments and is commonly observed in association with established filaments. Cancellation is interpreted as an important evolutionary change in the magnetic field that can lead to configurations suitable for the formation of filaments.

  13. Interaction of Two Filaments in a Long Filament Channel Associated with Twin Coronal Mass Ejections

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

    Zheng, Ruisheng; Chen, Yao; Wang, Bing

    Using the high-quality observations of the Solar Dynamics Observatory , we present the interaction of two filaments (F1 and F2) in a long filament channel associated with twin coronal mass ejections (CMEs) on 2016 January 26. Before the eruption, a sequence of rapid cancellation and emergence of the magnetic flux has been observed, which likely triggered the ascending of the west filament (F1). The east footpoints of rising F1 moved toward the east far end of the filament channel, accompanied by post-eruption loops and flare ribbons. This likely indicated a large-scale eruption involving the long filament channel, which resulted frommore » the interaction between F1 and the east filament (F2). Some bright plasma flew over F2, and F2 stayed at rest during the eruption, likely due to the confinement of its overlying lower magnetic field. Interestingly, the impulsive F1 pushed its overlying magnetic arcades to form the first CME, and F1 finally evolved into the second CME after the collision with the nearby coronal hole. We suggest that the interaction of F1 and the overlying magnetic field of F2 led to the merging reconnection that forms a longer eruptive filament loop. Our results also provide a possible picture of the origin of twin CMEs and show that the large-scale magnetic topology of the coronal hole is important for the eventual propagation direction of CMEs.« less

  14. Femtosecond Laser Filamentation for Atmospheric Sensing

    PubMed Central

    Xu, Huai Liang; Chin, See Leang

    2011-01-01

    Powerful femtosecond laser pulses propagating in transparent materials result in the formation of self-guided structures called filaments. Such filamentation in air can be controlled to occur at a distance as far as a few kilometers, making it ideally suited for remote sensing of pollutants in the atmosphere. On the one hand, the high intensity inside the filaments can induce the fragmentation of all matters in the path of filaments, resulting in the emission of characteristic fluorescence spectra (fingerprints) from the excited fragments, which can be used for the identification of various substances including chemical and biological species. On the other hand, along with the femtosecond laser filamentation, white-light supercontinuum emission in the infrared to UV range is generated, which can be used as an ideal light source for absorption Lidar. In this paper, we present an overview of recent progress concerning remote sensing of the atmosphere using femtosecond laser filamentation. PMID:22346566

  15. Powering of Hα Filaments by Cosmic Rays

    NASA Astrophysics Data System (ADS)

    Ruszkowski, Mateusz; Yang, H.-Y. Karen; Reynolds, Christopher S.

    2018-05-01

    Cluster cool cores possess networks of line-emitting filaments. These filaments are thought to originate via uplift of cold gas from cluster centers by buoyant active galactic nuclei (AGNs) bubbles, or via local thermal instability in the hot intracluster medium (ICM). Therefore, the filaments are either the signatures of AGN feedback or feeding of supermassive black holes. Despite being characterized by very short cooling times, the filaments are significant Hα emitters, which suggests that some process continuously powers these structures. Many cool cores host diffuse radio mini halos and AGN injecting radio plasma, suggesting that cosmic rays (CRs) and magnetic fields are present in the ICM. We argue that the excitation of Alfvén waves by CR streaming, and the replenishment of CR energy via accretion onto the filaments of high-plasma-β ICM characterized by low CR pressure support, can provide the adequate amount of heating to power and sustain the emission from these filaments. This mechanism does not require the CRs to penetrate the filaments, even if the filaments are magnetically isolated from the ambient ICM, and it may operate irrespectively of whether the filaments are dredged up from the center or form in situ in the ICM. This picture is qualitatively consistent with non-thermal line ratios seen in the cold filaments. Future X-ray observations of the iron line complex with XARM, Lynx, or Athena could help to test this model by providing constraints on the amount of CRs in the hot plasma that is cooling and accreting onto the filaments.

  16. Solar filament material oscillations and drainage before eruption

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

    Bi, Yi; Jiang, Yunchun; Yang, Jiayan

    Both large-amplitude longitudinal (LAL) oscillations and material drainage in a solar filament are associated with the flow of material along the filament axis, often followed by an eruption. However, the relationship between these two motions and a subsequent eruption event is poorly understood. We analyze a filament eruption using EUV imaging data captured by the Atmospheric Imaging Array on board the Solar Dynamics Observatory and the Hα images from the Global Oscillation Network Group. Hours before the eruption, the filament was activated, with one of its legs undergoing a slow rising motion. The asymmetric activation inclined the filament relative tomore » the solar surface. After the active phase, LAL oscillations were observed in the inclined filament. The oscillation period increased slightly over time, which may suggest that the magnetic fields supporting the filament evolve to be flatter during the slow rising phase. After the oscillations, a significant amount of filament material was drained toward one filament endpoint, followed immediately by the violent eruption of the filament. The material drainage may further support the change in magnetic topology prior to the eruption. Moreover, we suggest that the filament material drainage could play a role in the transition from a slow to a fast rise of the erupting filament.« less

  17. RadVel: General toolkit for modeling Radial Velocities

    NASA Astrophysics Data System (ADS)

    Fulton, Benjamin J.; Petigura, Erik A.; Blunt, Sarah; Sinukoff, Evan

    2018-01-01

    RadVel models Keplerian orbits in radial velocity (RV) time series. The code is written in Python with a fast Kepler's equation solver written in C. It provides a framework for fitting RVs using maximum a posteriori optimization and computing robust confidence intervals by sampling the posterior probability density via Markov Chain Monte Carlo (MCMC). RadVel can perform Bayesian model comparison and produces publication quality plots and LaTeX tables.

  18. Extracting BI-RADS Features from Portuguese Clinical Texts.

    PubMed

    Nassif, Houssam; Cunha, Filipe; Moreira, Inês C; Cruz-Correia, Ricardo; Sousa, Eliana; Page, David; Burnside, Elizabeth; Dutra, Inês

    2012-01-01

    In this work we build the first BI-RADS parser for Portuguese free texts, modeled after existing approaches to extract BI-RADS features from English medical records. Our concept finder uses a semantic grammar based on the BIRADS lexicon and on iterative transferred expert knowledge. We compare the performance of our algorithm to manual annotation by a specialist in mammography. Our results show that our parser's performance is comparable to the manual method.

  19. Prediction of Solar Eruptions Using Filament Metadata

    NASA Astrophysics Data System (ADS)

    Aggarwal, Ashna; Schanche, Nicole; Reeves, Katharine K.; Kempton, Dustin; Angryk, Rafal

    2018-05-01

    We perform a statistical analysis of erupting and non-erupting solar filaments to determine the properties related to the eruption potential. In order to perform this study, we correlate filament eruptions documented in the Heliophysics Event Knowledgebase (HEK) with HEK filaments that have been grouped together using a spatiotemporal tracking algorithm. The HEK provides metadata about each filament instance, including values for length, area, tilt, and chirality. We add additional metadata properties such as the distance from the nearest active region and the magnetic field decay index. We compare trends in the metadata from erupting and non-erupting filament tracks to discover which properties present signs of an eruption. We find that a change in filament length over time is the most important factor in discriminating between erupting and non-erupting filament tracks, with erupting tracks being more likely to have decreasing length. We attempt to find an ensemble of predictive filament metadata using a Random Forest Classifier approach, but find the probability of correctly predicting an eruption with the current metadata is only slightly better than chance.

  20. Core filaments of the nuclear matrix

    PubMed Central

    1990-01-01

    The nuclear matrix is concealed by a much larger mass of chromatin, which can be removed selectively by digesting nuclei with DNase I followed by elution of chromatin with 0.25 M ammonium sulfate. This mild procedure removes chromatin almost completely and preserves nuclear matrix morphology. The complete nuclear matrix consists of a nuclear lamina with an interior matrix composed of thick, polymorphic fibers and large masses that resemble remnant nucleoli. Further extraction of the nuclear matrices of HeLa or MCF-7 cells with 2 M sodium chloride uncovered a network of core filaments. A few dark masses remained enmeshed in the filament network and may be remnants of the nuclear matrix thick fibers and nucleoli. The highly branched core filaments had diameters of 9 and 13 nm measured relative to the intermediate filaments. They may serve as the core structure around which the matrix is constructed. The core filaments retained 70% of nuclear RNA. This RNA consisted both of ribosomal RNA precursors and of very high molecular weight hnRNA with a modal size of 20 kb. Treatment with RNase A removed the core filaments. When 2 M sodium chloride was used directly to remove chromatin after DNase I digestion without a preceding 0.25 M ammonium sulfate extraction, the core filaments were not revealed. Instead, the nuclear interior was filled with amorphous masses that may cover the filaments. This reflected a requirement for a stepwise increase in ionic strength because gradual addition of sodium chloride to a final concentration of 2 M without an 0.25 M ammonium sulfate extraction uncovered core filaments. PMID:2307700